GB virus C (hepatitis G virus) for the treatment of HIV

ABSTRACT

GB virus C (GBV-C or hepatitis G virus) is a flavivirus that frequently leads to chronic viremia in humans. The invention provides compositions and methods involving an anti-GBV-C antibody or other GBV-C binding agent, or a GBV-C antigen, for inhibiting and treating HIV infections.

BACKGROUND OF THE INVENTION

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/475,987, filed on Jun. 5, 2003, which is incorporated hereinby reference in its entirety.

The U.S. Government own rights in this invention pursuant to grantnumber R01AA12671 from the National Institutes of Health and a meritgrant awarded to Jack Stapleton from the Veterans Administration.

I. Field of the Invention

The present invention relates generally to the fields of molecularbiology and virology. More particularly, it concerns methods andcompositions to treat, inhibit or prevent HIV infection.

II. Description of Related Art

A. GB Virus Type C

GB virus type C (GBV-C), also known as hepatitis G virus (HGV), is avirus whose genomic organization and nucleotide sequence places it inthe Flavivirus family (Robertson et al., 1998). It is the most closelyrelated human virus to hepatitis C virus (HCV) (Leary et al., 1996;Linnen et al., 1996; Simons et al., 1995). It has been suggested thatthese viruses should be classified together with non-human GB-hepatitisagents as the hepacivirus genus. Although GBV-C was originallyassociated with post-transfusion hepatitis in humans (Linnen et al.,1996), subsequent epidemiological studies indicated that it does notcause acute or chronic hepatitis (Alter et al., 1997a; Alter et al.,1997b). In addition, experimental GBV-C infection of chimpanzees was notassociated with acute hepatitis (Bukh et al., 1998).

Persistent GBV-C viremia (as detected by RT-PCR) is common, with 0.9% to3% of healthy U.S. blood donors and approximately 20%-30% of patientswith HCV infection persistently infected with GBV-C (Dawson et al.,1996; Feucht et al., 1997; Simons et al., 1995a; Simons et al., 1995b;Tacke et al., 1997). Following infection, about 80% of people cleartheir viremia, concomitantly developing antibody to the GBV-C E2 protein(Feucht et al., 1997; Thomas et al., 1998). Thus, it is estimated thatapproximately 20% of infected people remain viremic for long periods oftime. GBV-C appears to be transmitted primarily by parenteral exposure(Simons et al., 1995), although there are data suggesting that sexualand/or household transmission of GBV-C infection may occur (Akiyoshi etal., 1999; de Martino et al., 1998; Nerurkar et al., 1998; Tanaka etal., 1997; Wu et al., 1997).

B. GBV-C and HIV

GBV-C has been investigated in the context of HIV infection. The courseof HIV-1 infection is extremely variable among infected individuals,although the reasons for this observation are not fully understood.Individuals whose HIV disease progresses slowly are often calledlong-term non-progressors (LTNPs). The prevalence of LTNPs varies from1% to 25% of infected people, depending upon the definition used(reviewed in Easterbrook, 1999). There are no specific clinical criteriafor LTNP. However, non-progression generally implies the absence ofHIV-related clinical disease 10 or more years following infection and anabsolute CD4 count of ≧500 cells/mm³ (Easterbrook, 1999). Evaluation ofLTNP's has identified HIV isolates with deletions in key replicativegenes (Deacon et al., 1995) and host genetic factors, including specificHLA haplotypes (reviewed in reference Rowland-Jones, 1999). In someindividuals, polymorphisms that result in absent or reduced expressionof HIV co-receptors have been identified (Huang et al., 1996). However,these findings are uncommon and thought to account for no more thanone-third of LTNP's (Rowland-Jones, 1999).

Persistent GBV-C infection is common in humans, with infection rates ofapproximately 0.9% to 3% in healthy blood donors, 20-30% in HCV-positivepeople (Dawson et al., 1996), and 35%-40% in HIV-positive individuals.GBV-C infection can persist for decades in the absence of any clinicalmorbidity or mortality. Among immune-competent individuals, it isestimated that 60% to 75% of GBV-C-infected people clear the infection,concomitantly developing antibodies to the envelope glycoprotein E2(Thomas et al., 1998). It is also known that GBV-C can be propagated incultures of peripheral blood mononuclear cells (PBMC's) (Fogeda et al.,1999).

In 1998, Toyoda et al. found that hemophiliacs co-infected with HIV andGBV-C had a lower plasma HIV RNA concentration and a lower incidence ofAIDS diagnoses compared to those infected with HIV alone (Toyoda et al.,1998), although the differences were not statistically significant. Incontrast, Sabin and colleagues found an increased rate of AIDS and deathin hemophiliacs “exposed” to GBV-C (Sabin et al., 1998) compared tonon-exposed individuals. This study included HIV-positive subjects whowere either GBV-C viremic as determined by detection of GBV-C RNA inplasma, or HIV-infected people who were not viremic but were anti-GBV-CE2 antibody-positive. Although the mortality rate was higher among theGBV-C “exposed” individuals, the results were not statisticallysignificant. Looking at HIV-infected persons, Lefrère and colleaguesreported a significant delay in the rate of CD4+ T cell decline,development of AIDS, and death in 23 HIV-positive individuals with GBV-Cviremia compared to 72 HIV-infected people without GBV-C viremia(Lefrère et al., 1999). In this study, HIV-infected individuals who werealso GBV-C-positive were compared to HIV-infected individuals who wereGBV-C-negative. When these subjects were matched by age, sex, baselineHIV RNA load, and baseline CD4 T cell count, HIV disease progressionappeared to be worse in GBV-C-negative subjects.

During progressive human immunodeficiency virus type 1 (HIV-1)infection, the virus-specific immune responses of an infected subjectgradually deteriorate, leading to the development of acquiredimmunodeficiency syndrome (AIDS). Most infected patients do not exhibitovert clinical manifestations of the disease for six to ten yearsfollowing initial infection, however, most individuals infected with HIVeventually die from conditions or infections; that the individual'simmune system is no longer equipped to fight. While treatment for AIDShas been forthcoming, no effective cure has been reported. Thus,preventative and treatment options against HIV infection and thedevelopment of AIDS remain highly desirable.

SUMMARY OF THE INVENTION

Embodiments of the invention provide improved methods and compositionsfor therapeutic and/or prophylatic treatment of HIV infection. Aspectsof the invention include compositions and methods related to antigensand/or polypeptides or peptides derived from GBV-C proteins or envelopeproteins, in particular GBV-C envelope protein E2 (E2). In otheraspects, the invention includes compositions and methods related toantibodies and other binding agents that bind antigens derived fromGBV-C proteins. In other aspects, the invention includes compositionsand methods related to the use of compositions comprising one or moreGBV-C polypeptides or peptides for therapeutic or prophylaticadministration. Compositions and methods comprising GBV-C polypeptides,GBV-C binding agents, or polynucleotides expressing the same can be usedto stimulate or provide anti-HIV activity, including, but not limited toinhibition of HIV replication, inhibition of HIV processing, HIVneutralization, inhibition of HIV infection, or a decreased or delayedmoratlity in infected persons.

Embodiments of the invention include a therapeutic compositioncomprising a GBV-C polypeptide or peptide, or a GBV-C polypeptide orpeptide binding agent, wherein the composition attenuates HIVinfectivity. The binding agent may be an antibody, an aptamer, or anyother known binding agent that can be selected or screened for bindingto GBV-C polypeptides or fragments thereof including, but not limited toGBV-C E2 polypeptides or peptides. In certain embodiments, the antibodymay be a polyclonal antibody, a monoclonal antibody or a fragment ormimetic thereof. An antibody of the invention may be a humanizedantibody, human antibody, or a human mouse, or human library derivedmonoclonal antibody. In certain embodiments, a GBV-C peptide can bederived from a GBV-C envelope protein. In particular embodiments, theGBV-C envelope protein is an E2 protein.

Embodiments of the invention include methods for preventing or treatingHIV infection comprising administering to a subject a compositioncomprising a GBV-C polypeptide or peptide-binding agent. The bindingagent may attenuate HIV infectivity. The binding agent may be anaptamer, an anti-GBV-C antibody, an antibody-like molecule, or otherknown binding agent that binds to a GBV-C polypeptide or peptide. Incertain embodiments, the anti-GBV-C binding agent binds to a GBV-C E2polypeptide or peptide. An anti-GBV-C antibody can be an anti-GBV-C E2antibody. An antibody of the invention may be a polyclonal, monoclonalor a fragment or mimetic therof. An antibody of the invention may be ahumanized antibody, human antibody, or a human mouse, or human libraryderived monoclonal antibody.

In certain embodiments, methods may include administration of at least asecond anti-HIV therapy. A second anti-HIV therapy may be anadministration of an infectious GBV-C virus, HAART therapy, AZT therapy,or other known HIV therapies. The second therapy may be administeredbefore, after or during a therapy comprising a GBV-C binding agent orGBV-C polypepitde or peptide. In a particular embodiment, a method mayinclude administering the GBV-C virus before a therapeutic compositionof the invention. A therapeutic composition of the invention may beadministered at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times overminutes, hours, days, weeks, months and/or years.

Embodiments of the invention include methods of preparing an antibody orother GBV-C binding agent by immunizing a non-human animal with a GBV-Cpolypeptide or fragment thereof, or a GBV-C E2 polypeptide or fragmentthereof, or screening recombinant human antibody libraries with theabove. In certain aspects, an antigen may be a GBV-C E2 derived peptide.In particular embodiments, the peptide may include, but is not limitedto, LTGGFYEPLVRRC (SEQ ID NO:6), GGAGLTGGFYEPLVRRC (SEQ ID NO:7), orFYEPLVRRC (SEQ ID NO:8).

Methods of preparing a therapeutic composition may comprise contacting acell with a polynucleotide encoding a GBV-C polypeptide or peptidebinding agent under conditions effective to allow expression of all orpart of a GBV-C polypeptide or peptide binding agent; collecting theexpressed GBV-C polypeptide or peptide binding agent; and constitutingthe GBV-C polypeptide or peptide binding agent in a pharmaceuticallyacceptable solution. The binding agent may attenuate, inhibit, and/ormodify HIV. A GBV-C polypeptide or peptide binding agent may be anaptamer, an antibody, or a related molecule. An antibody or relatedmolecule may be humanized.

Certain embodiments include vaccines comprising an antigen derived froma GBV-C polypeptide. The antigen may be all or part of a GBV-Cpolypeptide including, but not limited to a GBV-C E2 polypeptide. Incertain embodiments, the antigen may be a GBV-C E2-derived peptide. Inparticular embodiments, the peptide may include, but is not limited to,LTGGFYEPLVRRC (SEQ ID NO:6), or GGAGLTGGFYEPLVRRC (SEQ ID NO:7), orFYEPLVRRC (SEQ ID NO:8).

Embodiments of the invention include methods of immunizing a subjectcomprising contacting said subject with a composition comprising a GBV-Cpolypeptide or fragment thereof. The composition may further comprise anadjuvant. In certain embodiments, the GBV-C polypeptide is an E2polypeptide.

In still further embodiments, polypeptides and/or peptides of theinvention may be used as competititors for HIV binding to or associationwith various components of the human body.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” Furthermore, where multiple steps of amethod of process are cited, it is understood that the steps are notrequired to be performed in the particular order recited unless one ofskill in the art is not be able to practice the method in a differentorder.

Other objects, features, and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Exemplary effect of E2 antibody positive serum on HIVreplication.

FIG. 2. Example of inhibition of HIV by two E2 antibody positive sera ina dose-dependent fashion; whereas, E2 antibody negative sera does notinhibit HIV.

FIG. 3. IgG purified from E2 antibody positive sera inhibits HIVreplication in PBMC cultures.

FIG. 4. IgG purified from E2 antibody positive sera inhibits HIVreplication in PBMC cultures (same data shown in FIG. 3, but representsthe data as percent inhibition in HIV p24 Ag in culture supernatantfluids).

FIG. 5. GBV-C E2 antibody positive IgG inhibition of clinical X4 HIVstrain.

FIG. 6. The Roche M6 monoclonal antibody inhibits HIV-1 (R5 strain)

FIG. 7. The Biodesign (Saco, Me.) and Virostat (Portland, Me.)antibodies also inhibited HIV-1, although not as efficiently as M6.

FIG. 8. A map of the epitopes identified by the Roche mAbs as describedin Schmolke et al. (1998).

FIG. 9. A map of exemplary GBV-C epitopes (Peptide GGAGLTGGFY EPLVRRC(SEQ ID NO:6)).

FIG. 10. Illustrates inhibition of HIV-R5 by 17-mer Rabbit serum day 3.

FIG. 11. Illustrates inhibition of HIV (X4) by Rabbit anti-peptide IgGday 2.

FIG. 12. Illustrates inhibition of HIV (X4) by 17-mer Rabbit serum day3.

FIG. 13. Illustrates the elution profile of P24 antigen and CPMs.

FIG. 14. Metabolically ³⁵S-labeled HIV particles were partially purifiedby size-exclusion chromatography and incubated with either isotypecontrol (IC) or anti-E2 McAb (concentrations indicated on X-axis).HIV-IgG complexes were immuno-precipitated using staph protein A(Pansorbin). Data represent HIV cpm precipitated by M6 (over backgroundisotype control cpm).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Anti-retroviral medications suppress viral replication in HIV disease,yet they have failed to eradicate the virus from the body due to themulti-faceted nature of HIV infection, as well as the complexities ofthe immune system. Methods are being developed that both preventinfection and boost the immune system to keep it functioning at a levelwhere it can assist in fighting HIV infection.

Thus, embodiments of the invention provide additional methods andcompositions for therapeutic and/or prophylatic treatment of HIVinfection. Aspects of the invention include compositions and methodsrelated to antigens derived from GBV-C envelope proteins, in particularGBV-C envelope protein E2 (E2). In other aspects, the invention includescompositions and methods related to antibodies and other binding agentsthat bind antigens derived from GBV-C proteins. In particular, bindingagents, such as aptamers and anti-GBV-C antibodies that bind GBV-C E2proteins, are contemplated. In particular embodiments, a therapeuticGBV-C binding agent is contemplated for the treatment of HIV infection.Certain embodiments of the invention include combination treatments forHIV infection using compositions of the invention in combination withother anti-retroviral or HIV therapies.

The inventors have shown that HIV-infected subjects that are co-infectedwith GB virus C (GBV-C) typically have reduced mortality and slowerprogression to AIDS as compared to HIV-infected subjects without GBV-Cco-infection. Infection of peripheral blood mononuclear cells (PBMCs)with GBV-C and HIV results in inhibition of HIV-1 replication. GBV-Cinfection typically inhibits HIV by inducing β-chemokines and reducingexpression of the HIV co-receptor CCR5, explaining part of thebeneficial clinical findings of GBV-C on HIV disease progression.Antibodies directed to the GBV-C virus have been noted and are typicallyused as a diagnostic agents, with no therapeutic use having beenascribed to them. The inventors now describe a therapeutic use forantibodies and/or binding agents that bind GBV-C proteins (e.g.,envelope proteins), and antigens used for producing these antibodies orbinding agents. In certain embodiments, a vaccine composition includespeptides derived from GBV-C polypeptides. In further aspects, thepeptides themselves may bind to or associate with binding sites withinan organism that also bind to HIV, thus the peptides themselves may beused as competitive inhibitors of HIV binding or localization in a hostorganism.

Embodiments of the invention include anti-GBV-C antibodies that alsoattenuate the infectivity of HIV. Antibodies against the GBV-C envelopeglycoprotein E2 (GBV-C-E2), derived form either passive or activevaccination, are of particular interest for attenuation of HIV. Theinvention concerns the observation that antibodies against GBV-Cpeptides and polypeptides may react with and attenuate HIV. Theseantibodies may be induced or administered in a pharmaceuticalcomposition for the therapeutic or prophylatic treatment of HIVinfection. Infectious GBV-C can be used in combination with the presentinvention for preventative or therapeutic treatments for HIV infectionand related conditions such as AIDS.

I. GBV-C Virus

Like other members of the Flaviviridae, GBV-C is a positive-strand RNAvirus that encodes a single long open reading frame (Leary et al.,1996). GBV-C does not cause acute or chronic hepatitis, yet it is thefamily member most closely related to HCV, the cause of hepatitis C.Sequences of GBV-C have been previously reported, for example in U.S.Pat. No. 5,874,563, which is specifically incorporated by reference. Inparticular, an infectious GBV-C clone has been described in the PCTapplication WO 01/77157, which is incorporated herein by reference.

The GBV-C polyprotein is predicted to be cleaved into two envelopeproteins (E1 and E2, referred to collectively as GBV-C envelopeprotein), an RNA helicase, a trypsin-like serine protease, and anRNA-dependent RNA polymerase. A major difference between GBV-C and HCVis in the amino terminus of the polyprotein. In many isolates, thisregion is truncated, and no core (or nucleocapsid) protein is present(Simons et al., 1995; Xiang et al., 1999). In vitro translationexperiments suggest that the AUG immediately upstream of the putative E1protein is preferentially used to initiate translation, although theremay be as many as four AUG's in frame with the polyprotein upstream ofthis AUG (Simons et al., 1996).

The site of GBV-C replication has not been clearly identified, but itappears that replication in the hepatocyte, if it occurs, is not theprimary source of virus in infected individuals (Laskus et al., 1998;Pessoa et al., 1998; Seipp et al., 1999). Recently, there were reportsthat human peripheral blood mononuclear cells (PBMC's) andinterferon-resistant Daudi cells are permissive for GBV-C replication(Fogeda et al., 1999; Shimizu, 1999). In addition, transient replicationof GBV-C was described in MT-2 cells (a human T-cell line), and PH5CH (ahuman hepatocyte line immortalized with simian virus 40 large T antigen)(Seipp et al., 1999).

II. GBV-C Polypeptides

In certain aspects, the invention is directed to the function, activity,or antigenicity of various components of an infectious GBV-C virus or apolypeptide derived there from, in particular the E2 protein. Theexpression or isolation of certain GBV-C polypeptides can be used tostimulate an anti-HIV activity, including inhibition of replication,processing, neutralization, and infection. SEQ ID NO:2 and 4 representthe translated product of SEQ ID NO:1 (GBV-C polyprotein) and 3 (GBV-CE2 protein), respectively. It is contemplated that the compositions andmethods disclosed herein may be utilized to express all or part of SEQID NO:2 or 4 and derivates thereof. In certain embodiments, compositionsof the invention may include the nucleic acids encoding the peptides asset forth in SEQ ID NOs:5, 6, 7 or 8. Determination of which moleculespossess or stimulate an anti-HIV response may be achieved usingfunctional assays measuring HIV infectivity, which are familiar to thoseof skill in the art. In other embodiments of the invention, heterologouspolypeptides may be encoded by a sequence that also contains GBV-Csequences. “Heterologous” polypeptide indicates the polypeptide is not aGBV-C polypeptide. An endogenous GBV-C polypeptide refers to apolypeptide encoded by GBV-C viral RNA. Such a polypeptide would possessthe same or similar sequence as SEQ ID NO:2, 4, 5, 6, 7 or 8.

In certain embodiments, an antigen containing a 9 amino acid sequenceFYEPLVRRC (SEQ ID NO:8) or derivative thereof is contemplated. Incertain embodiments, an antigen comprising a 13 amino acid sequence(LTGGFYEPLVRRC, SEQ ID NO:6) or a derivative thereof is contemplated. Instill further embodiments, an antigen comprising a 17 amino acidsequence (GGAGLTGGFYEPLVRRC, SEQ ID NO:7) or derivative there of iscontemplated. The structure of the various peptides can be modeled orresolved by computer modeling, NMR, or x-ray crystallography. Peptidestructures may be used to engineer derivatives of the various E2 proteinsequences or to engineer other molecules to interact with the peptides,such as antibodies or other affinity reagents. Amino acids or peptidesof the invention may be used as an HIV disease-modifying immunogen(vaccine). Peptides may be used to inhibit, produce, or designinhibitors of HIV (as a prototype drug), as well as being used to induceanti-HIV antibodies (as a vaccine).

A. Variants of GBV-C Polypeptides

Embodiments of the invention include various GBV-C polypeptides,peptides, and derivatives thereof. Amino acid sequence variants of apolypeptide can be substitutional, insertional or deletion variants.Deletion variants lack one or more residues of the native protein thatare not essential for function or immunogenic activity. Insertionalmutants typically involve the addition of material at a non-terminalpoint in the polypeptide. This may include the insertion of animmunoreactive epitope or simply a single residue. Terminal additions,called fusion proteins, are discussed below.

Substitutional variants typically contain the exchange of one amino acidfor another at one or more sites within the protein, and may be designedto modulate one or more properties of the polypeptide, such as stabilityagainst proteolytic cleavage, without the loss of other functions orproperties. Substitutions of this kind preferably are conservative, thatis, one amino acid is replaced with one of similar shape and charge.Conservative substitutions are well known in the art and include, forexample, the changes of: alanine to serine; arginine to lysine;asparagine to glutamine or histidine; aspartate to glutamate; cysteineto serine; glutamine to asparagine; glutamate to aspartate; glycine toproline; histidine to asparagine or glutamine; isoleucine to leucine orvaline; leucine to valine or isoleucine; lysine to arginine; methionineto leucine or isoleucine; phenylalanine to tyrosine, leucine ormethionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine.

The term “biologically functional equivalent” is well understood in theart and is further defined in detail herein. Accordingly, sequences thathave between about 70% and about 80%; or more preferably, between about81% and about 90%; or even more preferably, between about 91% and about99%; of amino acids that are identical or functionally equivalent to theamino acids of GBV-C polypeptides, for example SEQ ID NO:2, 4, 5, 6, 7,or 8, provided the biological activity, e.g., immunogenicity, of theprotein or peptide is maintained.

The term “functionally equivalent codon” is used herein to refer tocodons that encode the same amino acid, such as the six codons forarginine or serine, and also refers to codons that encode biologicallyequivalent amino acids (see Table 1, below).

Certain embodiments of the invention include various peptides and/orfusion proteins of GBV-C polypeptides, in particular GBV-C E2 protein.For example, all or part of a GBV-C and/or a GBV-C E2 protein as setforth in SEQ ID NO:2, 4, 5, 6, 7 and/or 8 may be used in variousembodiments of the invention. In certain embodiments, a fragment of theE2 or other GBV-C protein may comprise, but is not limited to about 5,about 6, about 7, about 8, about 9, about 10, about 11, about 12, about13, about 14, about 15, about 16, about 17, about 18, about 19, about20, about 21, about 22, about 23, about 24, about 25, about 26, about27, about 28, about 29, about 30, about 31, about 32, about 33, about34, about 35, about 36, about 37, about 38, about 39, about 40, about41, about 42, about 43, about 44, about 45, about 46, about 47, about48, about 49, about 50, about 51, about 52, about 53, about 54, about55, about 56, about 57, about 58, about 59, about 60, about 61, about62, about 63, about 64, about 65, about 66, about 67, about 68, about69, about 70, about 71, about 72, about 73, about 74, about 75, about76, about 77, about 78, about 79, about 80, about 81, about 82, about83, about 84, about 85, about 86, about 87, about 88, about 89, about90, about 91, about 92, about 93, about 94, about 95, about 96, about97, about 98, about 99, about 100, about 110, about 120, about 130,about 140, about 150, about 160, about 170, about 180, about 190, about200, about 210, about 220, about 230, about 240, about 250, about 275,about 300, about 325, about 350, about 375, about 400, about 425, about450, about 475, about 500, about 525, about 550, about 575, about 600,about 625, about 650, about 675, about 700, about 725, about 750, about775, about 800, about 825, about 850, about 875, about 900, about 925,about 950, about 975, about 1000, about 1100, about 1200, about 1300,about 1400, about 1500, about 1750, about 2000, about 2250, about 2500,or greater amino acid molecule residues, and any range derivabletherein.

It also will be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ sequences, and yet still be essentially as setforth in one of the sequences disclosed herein, so long as the sequencemeets the criteria set forth above, including the maintenance ofbiological activity (e.g., immunogenicity) where protein expression isconcerned. The addition of terminal sequences particularly applies tonucleic acid sequences that may, for example, include various non-codingsequences flanking either of the 5′ or 3′ portions of the coding region.

The following is a discussion based upon changing of the amino acids ofa GBV-C polypeptide or peptide to create an equivalent, or even animproved, second-generation molecule. For example, certain amino acidsmay be substituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, antigen-binding regions of antibodies or binding siteson substrate molecules. Since it is the interactive capacity and natureof a protein that defines that protein's biological functional activity,certain amino acid substitutions can be made in a protein sequence, andin its underlying DNA or RNA coding sequence, and nevertheless produce aprotein with like properties. It is thus contemplated by the inventorsthat various changes may be made in the DNA or RNA sequences of genes orcoding regions without appreciable loss of their biological utility oractivity, as discussed herein. Table 1 shows the codons that encodeparticular amino acids. TABLE 1 CODON TABLE Amino Acids Codons AlanineAla A GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAUGlutamic acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly GGGA GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUULysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU MethionineMet M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCUGlutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU SerineSer S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine ValV GUA GUC GUG GUU Tryptophan Tyr W UGG Tyrosine Tyr Y UAC UAU

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982). It is accepted thatthe relative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

It also is understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

It is understood that an amino acid substituted for another having asimilar hydrophilicity value still produces a biologically equivalentand immunologically equivalent protein.

In certain embodiments, a GBV-C polypeptide may be a fusion protein.Fusion proteins may alter the characteristics of a given polypeptide,such antigenicity or purification characteristics. A fusion protein is aspecialized type of insertional variant. This molecule generally has allor a substantial portion of the native molecule, linked at the N- orC-terminus, to all or a portion of a second polypeptide. For example,fusions typically employ leader sequences from other species to permitthe recombinant expression of a protein in a heterologous host. Anotheruseful fusion includes the addition of an immunologically active domain,such as an antibody epitope, to facilitate purification of the fusionprotein. Inclusion of a cleavage site at or near the fusion junctionwill facilitate removal of the extraneous polypeptide afterpurification. Other useful fusions include linking of functionaldomains, such as active sites from enzymes such as a hydrolase,glycosylation domains, cellular targeting signals, or transmembraneregions.

B. In vitro Production of GBV-C or Anti-GBV-C Polypeptides or Peptides

Various types of expression vectors are known in the art that can beused for the production of protein products. Following transfection witha expression vector, a cell in culture, e.g., a primary mammalian cell,a recombinant product may be prepared in various ways. A host cellstrain may be chosen that modulates the expression of the insertedsequences, or that modifies and processes the gene product in the mannerdesired. Such modifications (e.g., glycosylation) and processing (e.g.,cleavage) of protein products may be important for the function of theprotein. Different host cells have characteristic and specificmechanisms for the post-translational processing and modification ofproteins. Appropriate cell lines or host systems can be chosen to insurethe correct modification and processing of the foreign proteinexpressed. In order for the cells to be kept viable while in vitro andin contact with the expression construct, it is necessary to ensure thatthe cells maintain contact with the correct ratio of oxygen and carbondioxide and nutrients but are protected from microbial contamination.Cell culture techniques are well documented (for exemplary methods seeFreshney, 1992).

Animal cells can be propagated in vitro in two modes: asnon-anchorage-dependent cells growing in suspension throughout the bulkof the culture or as anchorage-dependent cells requiring attachment to asolid substrate for their propagation (i.e., a monolayer type of cellgrowth).

Non-anchorage dependent or suspension cultures from continuousestablished cell lines are the most widely used means of large-scaleproduction of cells and cell products. However, suspension culturedcells have limitations, such as tumorigenic potential and lower proteinproduction than adherent cells.

In further aspects of the invention, other protein production methodsknown in the art may be used, including but not limited to prokaryotic,yeast, and other eukaryotic hosts such as insect cells and the like.

C. Protein Purification

It may be desirable to purify anti-GBV-C and/or GBV-C polypeptides andpeptides, or variants and derivatives thereof. Protein purificationtechniques are well known to those of skill in the art. These techniquesinvolve, at one level, the crude fractionation of the cellular milieu topolypeptide and non-polypeptide fractions. Having separated thepolypeptide from other proteins, the polypeptide of interest may befurther purified using chromatographic and electrophoretic techniques toachieve partial or complete purification (or purification tohomogeneity). Analytical methods particularly suited to the preparationof a pure peptide are ion-exchange chromatography, hydrophobicinteraction chromatography, exclusion chromatography; polyacrylamide gelelectrophoresis; isoelectric focusing. A particularly efficient methodof purifying peptides is fast protein liquid chromatography or evenFPLC.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of an encodedprotein or peptide. The term “purified protein or peptide” as usedherein, is intended to refer to a composition, isolatable from othercomponents, wherein the protein or peptide is purified to any degreerelative to its naturally obtainable state. A purified protein orpeptide therefore also refers to a protein or peptide, free from theenvironment in which it may naturally occur.

Generally, “purified” will refer to a protein or peptide compositionthat has been subjected to fractionation to remove various othercomponents, and which composition substantially retains its expressedbiological activity. Where the term “substantially purified” is used,this designation will refer to a composition in which the protein orpeptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity, hereinassessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification and whetheror not the expressed protein or peptide exhibits a detectable activity.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme.

III. GBV-C Polynucleotides

Certain embodiments of the invention include GBV-C polynucleotides ornucleic acid molecules and fragments thereof. The polynucleotides of theinvention may be isolated and purified from GBV-C virus or cellsinfected or transfected with GBV-C polynucleotides. The term isolatedindicating they are free or substantially free from total viral orcellular genomic RNA or DNA, and proteins. It is contemplated that anisolated and purified GBV-C nucleic acid molecule may take the form ofRNA or DNA. A GBV-C nucleic acid molecule refers to an RNA or DNAmolecule that is capable of yielding all or part of a GBV-C polyproteinfrom a transfected cell.

As used in this application, the term “polynucleotide” refers to anucleic acid molecule, RNA, or DNA that has been isolated free of totalgenomic nucleic acid. Therefore, a “polynucleotide encoding all or partof GBV-C” refers to a nucleic acid segment that contains GBV-C codingsequences, yet is isolated away from, or purified and free of, totalviral genomic RNA and proteins; similarly, a “polynucleotide encodingfull-length GBV-C” refers to a nucleic acid segment that containsfull-length GBV-C coding sequences yet is isolated away from, orpurified and free of, total viral genomic RNA and protein. Therefore,when the present application refers to the function or activity of aninfectious GBV-C that is encoded by a GBV-C polynucleotide, it is meantthat the polynucleotide encodes a molecule that has the ability topropagate an infectious GBV-C virus particle from a cell. It iscontemplated that a GBV-C polynucleotide may refer to a GBV-C RNAtranscript that is able to propagate an infectious GBV-C virus particleafter introduction to a cell or to a GBV-C expression construct, clone,or vector composed of double-stranded DNA or DNA/RNA hybrid that issimilarly capable.

The term “cDNA” is intended to refer to DNA prepared using RNA as atemplate. The advantage of using a cDNA, as opposed to genomic RNA or anRNA transcript is stability and the ability to manipulate the sequenceusing recombinant DNA technology (See Maniatis, 1989; Ausubel, 1994).There may be times when the full or partial genomic sequence ispreferred. Alternatively, cDNAs may be advantageous because itrepresents coding regions of a polypeptide and eliminates introns andother regulatory regions.

It also is contemplated that a given GBV-C may be represented by naturalvariants or strains that have slightly different nucleic acid sequencesbut, nonetheless, encode the same viral polypeptides (see Table 1above). Consequently, the present invention also encompasses derivativesof GBV-C with minimal amino acid changes in its viral proteins, but thatpossesses the same activities.

The term “gene” is used for simplicity to refer to the nucleic acidgiving rise to a functional protein, polypeptide, or peptide-encodingunit. As will be understood by those in the art, this functional termincludes genomic sequences, cDNA sequences, and smaller engineered genesegments that express, or may be adapted to express, proteins,polypeptides, domains, peptides, fusion proteins, and mutants. Thenucleic acid molecule encoding GBV-C may contain a contiguous nucleicacid sequence encoding one or more GBV-C genes and regulatory regionsand be of the following lengths: about 10, 20, 30, 40, 50, 60, 70, 80,90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360,370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490,500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630,640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770,780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910,920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040,1050, 1060, 1070, 1080, 1090, 1100, 1200, 1300, 1400, 1500, 1600, 1700,1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900,3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100,4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300,5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500,6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700,7800, 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900,9000, 9100, 9200, 9300, 9400, 9500, 9600, 9700, 9800, 9900, 10000,10100, 10200, 10300, 10400, 10500, 10600, 10700, 10800, 10900, 11000,11100, 11200, 11300, 11400, 11500, 11600, 11700, 11800, 11900, 12000 ormore nucleotides, nucleosides, or base pairs. Such sequences may beidentical or complementary to all or part of SEQ ID NO:1, 3 or GenbankAccession numbers AY196904 or AF070476 or segments thereof, e.g., thosesegments related to peptides of SEQ ID NO:5, 6, 7 or 8.

“Isolated substantially away from other coding sequences” means that thegene of interest forms part of the coding region of the nucleic acidsegment, and that the segment does not contain large portions ofnaturally-occurring coding nucleic acid, such as large chromosomalfragments or other functional genes or cDNA coding regions. Of course,this refers to the nucleic acid segment as originally isolated, and doesnot exclude genes or coding regions later added to the segment by humanmanipulation.

In particular embodiments, the invention concerns isolated nucleic acidsegments and recombinant vectors incorporating DNA sequences that encodeGBV-C polypeptides or peptides that include within its amino acidsequence a contiguous amino acid sequence in accordance with, oressentially corresponding to GBV-C polypeptides.

Certain embodiments include nucleic acids segments and recombinantvectors encoding polypeptides and peptides to induce or enhance immuneresponses in both subjects having HIV, suspected of having HIV, at riskof being exposed to HIV and/or animals or cells for the production ofanti-GBV-C antibodies.

The nucleic acid segments used in the present invention, regardless ofthe length of the coding sequence itself, may be combined with other DNAor RNA sequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, other coding segments,and the like, such that their overall length may vary considerably. Itis therefore contemplated that a nucleic acid fragment of almost anylength may be employed, with the total length preferably being limitedby the ease of preparation and use in the intended recombinant DNAprotocol.

In a non-limiting example, one or more nucleic acid constructs may beprepared that include a contiguous stretch of nucleotides identical toor complementary to GBV-C. A nucleic acid construct may be about 50, 60,70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000,3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000,13,000, 14,000, 15,000, 20,000, 30,000, 50,000, 100,000, 250,000, about500,000, 750,000, to about 1,000,000 nucleotides in length, as well asconstructs of greater size, up to and including chromosomal sizes(including all intermediate lengths and intermediate ranges), given theadvent of nucleic acids constructs such as a yeast artificial chromosomeare known to those of ordinary skill in the art. It will be readilyunderstood that “intermediate lengths” and “intermediate ranges,” asused herein, means any length or range including or between the quotedvalues (i.e., all integers including and between such values).Non-limiting examples of intermediate lengths include about 11, about12, about 13, about 16, about 17, about 18, about 19, etc.; about 21,about 22, about 23, etc.; about 31, about 32, etc.; about 51, about 52,about 53, etc.; about 101, about 102, about 103, etc.; about 151, about152, about 153, etc.

The nucleic acid segments used in the present invention encompassbiologically functional and/or immunogenically equivalent GBV-C proteinsand peptides. Such sequences may arise as a consequence of codonredundancy and functional equivalency that are known to occur naturallywithin nucleic acid sequences and the proteins thus encoded.Alternatively, functionally and immunologically equivalent proteins orpeptides may be created via the application of recombinant DNAtechnology, in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged. Changes designed by human may be introduced through theapplication of site-directed mutagenesis techniques, e.g., to introduceimprovements to the antigenicity of the protein.

A. Vectors Encoding GBV-C

The present invention encompasses the use of vectors to encode for allor part of one or more GBV-C polypeptides, including an infectiousGBV-C. The term “vector” is used to refer to a carrier nucleic acidmolecule into which a nucleic acid sequence can be inserted forintroduction into a cell where it can be replicated. A nucleic acidsequence can be “exogenous,” which means that it is foreign to the cellinto which the vector is being introduced or that the sequence ishomologous to a sequence in the cell but in a position within the hostcell nucleic acid in which the sequence is ordinarily not found. Vectorsinclude plasmids, cosmids, viruses (bacteriophage, animal viruses, andplant viruses), and artificial chromosomes (e.g., YACs). In particularembodiments, gene therapy or immunization vectors are contemplated. Oneof skill in the art would be well equipped to construct a vector throughstandard recombinant techniques, which are described in Maniatis et al.,1988 and Ausubel et al., 1994, both incorporated herein by reference.

The term “expression vector” or “expression construct” refers to avector containing a nucleic acid sequence coding for at least part of agene product capable of being transcribed. In some cases, RNA moleculesare then translated into a protein, polypeptide, or peptide. In othercases, these sequences are not translated, for example, in theproduction of antisense molecules or ribozymes. Expression vectors cancontain a variety of “control sequences,” which refer to nucleic acidsequences necessary for the transcription and possibly translation of anoperably linked coding sequence in a particular host organism. Inaddition to control sequences that govern transcription and translation,vectors and expression vectors may contain nucleic acid sequences thatserve other functions as well and are described infra. It iscontemplated that an infectious GBV-C particle of the present inventionmay arise from a vector containing GBV-C sequence or RNA encoding GBV-Csequence into a cell. Either of these, or any other nucleic acidmolecules of the present invention may be constructed with any of thefollowing nucleic acid control sequences. Thus, the full-length RNAtranscript may contain the benefit of recombinant DNA technology suchthat it contains exogenous control sequences or genes.

1. Promoters and Enhancers

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind such as RNA polymerase and other transcriptionfactors. The phrases “operatively positioned,” “operatively linked,”“under control,” and “under transcriptional control” means that apromoter is in a correct functional location and/or orientation inrelation to a nucleic acid sequence to control transcriptionalinitiation and/or expression of that sequence. A promoter may or may notbe used in conjunction with an “enhancer,” which refers to a cis-actingregulatory sequence involved in the transcriptional activation of anucleic acid sequence.

A promoter may be one naturally associated with a gene or sequence, asmay be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other prokaryotic, viral, or eukaryotic cell, andpromoters or enhancers not “naturally occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression. In addition to producing nucleicacid sequences of promoters and enhancers synthetically, sequences maybe produced using recombinant cloning and/or nucleic acid amplificationtechnology, including PCR™, in connection with the compositionsdisclosed herein (see U.S. Pat. No. 4,683,202 and U.S. Pat. No.5,928,906, each incorporated herein by reference). Furthermore, it iscontemplated the control sequences that direct transcription and/orexpression of sequences within non-nuclear organelles such asmitochondria, chloroplasts, and the like, can be employed as well.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the nucleic acid segment inthe cell type, organelle, and organism chosen for expression. Those ofskill in the art of molecular biology generally know the use ofpromoters, enhancers, and cell type combinations for protein expression,for example, see Sambrook et al. (1989), incorporated herein byreference. The promoters employed may be constitutive, tissue-specific,inducible, and/or useful under the appropriate conditions to direct highlevel expression of the introduced DNA segment, such as is advantageousin the large-scale production of recombinant proteins and/or peptides.The promoter may be heterologous or exogenous, i.e., from a differentsource than GBV-C sequence. In some examples, a prokaryotic promoter isemployed for use with in vitro transcription of a desired sequence.Prokaryotic promoters for use with many commercially available systemsinclude T7, T3, and Sp6.

Table 2 lists several elements/promoters that may be employed, in thecontext of the present invention, to regulate the expression of a gene.This list is not intended to be exhaustive of all the possible elementsinvolved in the promotion of expression but, merely, to be exemplarythereof. Table 3 provides examples of inducible elements, which areregions of a nucleic acid sequence that can be activated in response toa specific stimulus. TABLE 2 Promoter and/or Enhancer Promoter/EnhancerReferences Immunoglobulin Heavy Chain Banerji et al., 1983; Gilles etal., 1983; Grosschedl et al., 1985; Atchinson et al., 1986, 1987; Imleret al., 1987; Weinberger et al., 1984; Kiledjian et al., 1988; Porton etal.; 1990 Immunoglobulin Light Chain Queen et al., 1983; Picard et al.,1984 T-Cell Receptor Luria et al., 1987; Winoto et al., 1989; Redondo etal.; 1990 HLA DQ a and/or DQ β Sullivan et al., 1987 β-InterferonGoodbourn et al., 1986; Fujita et al., 1987; Goodbourn et al., 1988Interleukin-2 Greene et al., 1989 Interleukin-2 Receptor Greene et al.,1989; Lin et al., 1990 MHC Class II 5 Koch et al., 1989 MHC Class IIHLA-DRa Sherman et al., 1989 β-Actin Kawamoto et al., 1988; Ng et al.;1989 Muscle Creatine Kinase (MCK) Jaynes et al., 1988; Horlick et al.,1989; Johnson et al., 1989 Prealbumin (Transthyretin) Costa et al., 1988Elastase I Omitz et al., 1987 Metallothionein (MTII) Karin et al., 1987;Culotta et al., 1989 Collagenase Pinkert et al., 1987; Angel et al.,1987 Albumin Pinkert et al., 1987; Tronche et al., 1989, 1990α-Fetoprotein Godbout et al., 1988; Campere et al., 1989 γ-Globin Bodineet al., 1987; Perez-Stable et al., 1990 β-Globin Trudel et al., 1987c-fos Cohen et al., 1987 c-HA-ras Triesman, 1986; Deschamps et al., 1985Insulin Edlund et al., 1985 Neural Cell Adhesion Molecule Hirsh et al.,1990 (NCAM) α₁-Antitrypain Latimer et al., 1990 H2B (TH2B) Histone Hwanget al., 1990 Mouse and/or Type I Collagen Ripe et al., 1989Glucose-Regulated Proteins Chang et al., 1989 (GRP94 and GRP78) RatGrowth Hormone Larsen et al., 1986 Human Serum Amyloid A (SAA) Edbrookeet al., 1989 Troponin I (TN I) Yutzey et al., 1989 Platelet-DerivedGrowth Factor Pech et al., 1989 (PDGF) Duchenne Muscular DystrophyKlamut et al., 1990 SV40 Banerji et al., 1981; Moreau et al., 1981;Sleigh et al., 1985; Firak et al., 1986; Herr et al., 1986; Imbra etal., 1986; Kadesch et al., 1986; Wang et al., 1986; Ondek et al., 1987;Kuhl et al., 1987; Schaffner et al., 1988 Polyoma Swartzendruber et al.,1975; Vasseur et al., 1980; Katinka et al., 1980, 1981; Tyndell et al.,1981; Dandolo et al., 1983; de Villiers et al., 1984; Hen et al., 1986;Satake et al., 1988; Campbell and/or Villarreal, 1988 RetrovirusesKriegler et al., 1982, 1983; Levinson et al., 1982; Kriegler et al.,1983, 1984a, b, 1988; Bosze et al., 1986; Miksicek et al., 1986;Celander et al., 1987; Thiesen et al., 1988; Celander et al., 1988; Cholet al., 1988; Reisman et al., 1989 Papilloma Virus Campo et al., 1983;Lusky et al., 1983; Spandidos and/or Wilkie, 1983; Spalholz et al.,1985; Lusky et al., 1986; Cripe et al., 1987; Gloss et al., 1987;Hirochika et al., 1987; Stephens et al., 1987; Glue et al., 1988Hepatitis B Virus Bulla et al., 1986; Jameel et al., 1986; Shaul et al.,1987; Spandau et al., 1988; Vannice et al., 1988 Human ImmunodeficiencyVirus Muesing et al., 1987; Hauber et al., 1988; Jakobovits et al.,1988; Feng et al., 1988; Takebe et al., 1988; Rosen et al., 1988;Berkhout et al., 1989; Laspia et al., 1989; Sharp et al., 1989; Braddocket al., 1989 Cytomegalovirus (CMV) Weber et al., 1984; Boshart et al.,1985; Foecking et al., 1986 Gibbon Ape Leukemia Virus Holbrook et al.,1987; Quinn et al., 1989

TABLE 3 Inducible Elements Element Inducer References MT II PhorbolEster (TFA) Palmiter et al., 1982; Heavy metals Haslinger et al., 1985;Searle et al., 1985; Stuart et al., 1985; Imagawa et al., 1987, Karin etal., 1987; Angel et al., 1987b; McNeall et al., 1989 MMTV (mouseGlucocorticoids Huang et al., 1981; Lee et mammary tumor al., 1981;Majors et al., virus) 1983; Chandler et al., 1983; Lee et al., 1984;Ponta et al., 1985; Sakai et al., 1988 β-Interferon poly(rI)x Tavernieret al., 1983 poly(rc) Adenovirus 5 E2 ElA Imperiale et al., 1984Collagenase Phorbol Ester (TPA) Angel et al., 1987a Stromelysin PhorbolEster (TPA) Angel et al., 1987b SV40 Phorbol Ester (TPA) Angel et al.,1987b Murine MX Gene Interferon, Hug et al., 1988 Newcastle DiseaseVirus GRP78 Gene A23187 Resendez et al., 1988 α-2-Macroglobulin IL-6Kunz et al., 1989 Vimentin Serum Rittling et al., 1989 MHC Class I GeneInterferon Blanar et al., 1989 H-2κb HSP70 ElA, SV40 Large T Taylor etal., 1989, 1990a, Antigen 1990b Proliferin Phorbol Ester-TPA Mordacq etal., 1989 Tumor Necrosis PMA Hensel et al., 1989 Factor ThyroidStimulating Thyroid Hormone Chatterjee et al., 1989 Hormone α Gene

The identity of tissue-specific promoters or elements, as well as assaysto characterize their activity, is well known to those of skill in theart. Examples of such regions include the human LIMK2 gene (Nomoto etal. 1999), the somatostatin receptor 2 gene (Kraus et al., 1998), murineepididymal retinoic acid-binding gene (Lareyre et al., 1999), human CD4(Zhao-Emonet et al., 1998), mouse alpha2 (XI) collagen (Tsumaki, et al.,1998), D1A dopamine receptor gene (Lee, et al., 1997), insulin-likegrowth factor II (Wu et al., 1997), human platelet endothelial celladhesion molecule-1 (Almendro et al., 1996).

2. Initiation Signals and Internal Ribosome Binding Sites

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

In certain embodiments of the invention, the use of internal ribosomeentry sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass theribosome-scanning model of 5′ methylated Cap dependent translation andbegin translation at internal sites (Pelletier and Sonenberg, 1988).IRES elements from two members of the picornavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message (see U.S. Pat.Nos. 5,925,565 and 5,935,819, herein incorporated by reference).

3. Multiple Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleicacid region that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector. (See Carbonelli et al., 1999, Levenson et al., 1998,and Cocea, 1997, incorporated herein by reference.) “Restriction enzymedigestion” refers to catalytic cleavage of a nucleic acid molecule withan enzyme that functions only at specific locations in a nucleic acidmolecule. Many of these restriction enzymes are commercially available.Use of such enzymes is widely understood by those of skill in the art.Frequently, a vector is linearized or fragmented using a restrictionenzyme that cuts within the MCS to enable exogenous sequences to beligated to the vector. “Ligation” refers to the process of formingphosphodiester bonds between two nucleic acid fragments, which may ormay not be contiguous with each other. Techniques involving restrictionenzymes and ligation reactions are well known to those of skill in theart of recombinant technology.

4. Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing toremove introns from the primary transcripts. Vectors containing genomiceukaryotic sequences may require donor and/or acceptor splicing sites toensure proper processing of the transcript for protein expression. (SeeChandler et al., 1997, herein incorporated by reference.)

5. Termination Signals

The vectors or constructs of the present invention will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the DNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase. Thus, in certainembodiments a termination signal that ends the production of an RNAtranscript is contemplated. A terminator may be necessary in vivo toachieve desirable message levels.

In eukaryotic systems, the terminator region may also comprise specificDNA sequences that permit site-specific cleavage of the new transcriptto expose a polyadenylation site. This signals a specialized endogenouspolymerase to add a stretch of about 200 A residues (polyA) to the 3′end of the transcript. RNA molecules modified with this polyA tailappear to more stable and are translated more efficiently. Thus, inother embodiments involving eukaryotes, it is preferred that thatterminator comprises a signal for the cleavage of the RNA, and it ismore preferred that the terminator signal promotes polyadenylation ofthe message. The terminator and/or polyadenylation site elements canserve to enhance message levels and/or to minimize read through from thecassette into other sequences.

Terminators contemplated for use in the invention include any knownterminator of transcription described herein or known to one of ordinaryskill in the art, including but not limited to, for example, thetermination sequences of genes, such as for example the bovine growthhormone terminator or viral termination sequences, such as for examplethe SV40 terminator. In certain embodiments, the termination signal maybe a lack of transcribable or translatable sequence, such as due to asequence truncation.

6. Polyadenylation Signals

For expression, particularly eukaryotic expression, one will typicallyinclude a polyadenylation signal to effect proper polyadenylation of thetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and/or any suchsequence may be employed. Preferred embodiments include the SV40polyadenylation signal and/or the bovine growth hormone polyadenylationsignal, convenient and/or known to function well in various targetcells. Polyadenylation may increase the stability of the transcript ormay facilitate cytoplasmic transport.

7. Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively, an autonomously replicating sequence (ARS) can beemployed if the host cell is yeast.

8. Selectable and Screenable Markers

In certain embodiments of the invention, the cells containing a nucleicacid construct of the present invention may be identified in vitro or invivo by including a marker in the expression vector. Such markers wouldconfer an identifiable change to the cell permitting easy identificationof cells containing the expression vector. Generally, a selectablemarker is one that confers a property that allows for selection. Apositive selectable marker is one in which the presence of the markerallows for its selection, while a negative selectable marker is one inwhich its presence prevents its selection. An example of a positiveselectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscalorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

B. Host Cells

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which refers to any and all subsequent generations. It is understoodthat all progeny may not be identical due to deliberate or inadvertentmutations. In the context of expressing a heterologous nucleic acidsequence, “host cell” refers to a prokaryotic or eukaryotic cell, and itincludes any transformable organisms that is capable of replicating avector and/or expressing a heterologous gene encoded by a vector. A hostcell can, and has been, used as a recipient for vectors. A host cell maybe “transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid is transferred or introduced into the host cell.A transformed cell includes the primary subject cell and its progeny.

Host cells may be derived from prokaryotes or eukaryotes, depending uponwhether the desired result is replication of the vector, expression ofpart or all of the vector-encoded nucleic acid sequences, or productionof infectious viral particles. Numerous cell lines and cultures areavailable for use as a host cell, and they can be obtained through theAmerican Type Culture Collection (ATCC), which is an organization thatserves as an archive for living cultures and genetic materials. Anappropriate host can be determined by one of skill in the art based onthe vector backbone and the desired result. A plasmid or cosmid, forexample, can be introduced into a prokaryote host cell for replicationof many vectors. Bacterial cells used as host cells for vectorreplication and/or expression include DH5α, JM109, and KC8, as well as anumber of commercially available bacterial hosts such as SURE™ CompetentCells and SOLOPACK™ Gold Cells (STRATAGENE®, La Jolla). Alternatively,bacterial cells such as E. coli LE392 could be used as host cells forphage viruses.

Examples of eukaryotic host cells for replication and/or expression of avector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Manyhost cells from various cell types and organisms are available and wouldbe known to one of skill in the art. Similarly, a viral vector may beused in conjunction with either an eukaryotic or prokaryotic host cell,particularly one that is permissive for replication or expression of thevector.

C. Expression Systems

Numerous expression systems exist that comprise at least all or part ofthe compositions discussed above. Prokaryote- and/or eukaryote-basedsystems can be employed for use with the present invention to producenucleic acid sequences, or their cognate polypeptides, proteins andpeptides. Many such systems are commercially and widely available.

The insect cell/baculovirus system can produce a high level of proteinexpression of a heterologous nucleic acid segment, such as described inU.S. Pat. Nos. 5,871,986 and 4,879,236, both herein incorporated byreference, and which can be bought, for example, under the name MAXBAC®2.0 from INVITROGEN® and BACPACK™ BACULOVIRUS EXPRESSION SYSTEM fromCLONTECH®.

Other examples of expression systems include STRATAGENE®'S COMPLETECONTROL™ Inducible Mammalian Expression System, which involves asynthetic ecdysone-inducible receptor, or its pET Expression System, anE. coli expression system. Another example of an inducible expressionsystem is available from INVITROGEN®, which carries the T-REX™(tetracycline-regulated expression) System, an inducible mammalianexpression system that uses the full-length CMV promoter. The Tet-On™and Tet-Off™ systems from CLONTECH® can be used to regulate expressionin a mammalian host using tetracycline or its derivatives. Theimplementation of these systems is described in Gossen et al., 1992 andGossen et al., 1995, and U.S. Pat. No. 5,650,298, all of which areincorporated by reference.

INVITROGEN® also provides a yeast expression system called the Pichiamethanolica Expression System, which is designed for high-levelproduction of recombinant proteins in the methylotrophic yeast Pichiamethanolica. One of skill in the art would know how to express a vector,such as an expression construct, to produce a nucleic acid sequence orits cognate polypeptide, protein, or peptide.

D. Introduction of Nucleic Acids into Cells

In certain embodiments, a nucleic acid may be introduce into a cell invitro for production of polypeptides or in vivo for immunizationpurposes. There are a number of ways in which nucleic acid moleculessuch as expression vectors may be introduced into cells. In certainembodiments of the invention, the expression vector comprises a GBV-Cinfectious particle or engineered vector derived from a GBV-C genome. Inother embodiments, an expression vector known to one of skill in the artmay be used to express a segment of a GBV-C nucleic, which may betranslated into a GBV-C polypeptide or peptide. The ability of certainviruses to enter cells via receptor-mediated endocytosis, to integrateinto host cell genome and express viral genes stably and efficientlyhave made them attractive candidates for the transfer of foreign genesinto mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein, 1988;Baichwal and Sugden, 1986; Temin, 1986).

“Viral expression vector” is meant to include those vectors containingsequences of that virus sufficient to (a) support packaging of thevector and (b) to express a polynucleotide that has been cloned therein.In this context, expression may require that the gene product besynthesized. A number of such viral vectors have already been thoroughlyresearched, including adenovirus, adeno-associated viruses,retroviruses, herpesviruses, and vaccinia viruses.

Delivery may be accomplished in vitro, as in laboratory procedures fortransforming cells lines, or in vivo or ex vivo, as in the treatment ofcertain disease states. One mechanism for delivery is via viralinfection where the expression vector is encapsidated in an infectiousviral particle. Several non-viral methods for the transfer of expressionvectors into cultured mammalian cells also are contemplated by thepresent invention. These include calcium phosphate precipitation (Grahamand Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990)DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al., 1986;Potter et al., 1984), direct microinjection (Harland and Weintraub,1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al.,1979) and lipofectamine-DNA complexes, cell sonication (Fechheimer etal., 1987), gene bombardment using high velocity microprojectiles (Yanget al., 1990), liposome (Ghosh and Bachhawat, 1991; Kaneda et al., 1989)and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988).Some of these techniques may be successfully adapted for in vivo or exvivo use.

In certain embodiments, the nucleic acid encoding a gene or genes may bestably integrated into the genome of the cell. This integration may bein the cognate location and orientation via homologous recombination(gene replacement) or it may be integrated in a random, non-specificlocation (gene augmentation). In yet further embodiments, the nucleicacid may be stably maintained in the cell as a separate, episomalsegment of DNA. Such nucleic acid segments or “episomes” encodesequences sufficient to permit maintenance and replication independentof or in synchronization with the host cell cycle. How the expressionvector is delivered to a cell and where in the cell the nucleic acidremains is dependent on the type of expression vector employed.

Transfer of a nucleic acid molecule may be performed by any of themethods mentioned above which physically or chemically permeabilize thecell membrane. This is particularly applicable for transfer in vitro,but it may be applied to in vivo use as well.

IV. GBV-C Related Immunotherapy

Embodiments of the invention include various compositions and methodsfor stimulating, supplementing or enhancing the immune system of asubject that has or may be exposed to HIV. Immunotherapy in general is atreatment to stimulate, enhance, or restore the ability of the immunesystem to fight infection and disease. Immunotherapy is thus any form oftreatment that uses the immune system to fight infection and disease orto protect the body from some of the side effects of treatment. Examplesinclude active immunization, passive immunization, and adoptiveimmunotherapies.

Immunoglobulins typically mediate humoral immunity by attaching toforeign antigens and activating effector modalities (e.g., complement,granulocytes, cytotoxic T-cells, etc.) to destroy and clear the antigensand also by passive inactivation, exclusion or immobilization ofpathogens. Each of the five Ig isotypes possesses its own spectrum ofeffector systems with which it interacts via its Fc domain. The constantregion isotype of the antibody is determined following T-cell mediated,Ig class-switching which endows a given antibody with the specificeffector modalities of the new isotype. Administration and/orelicitation of antibodies to GBV-C derived peptides, in particular GBV-CE2, may be used as a therapeutic in various immunotherapies.

A. Passive Immunotherapy Related to Anti-GBV-C Antibodies or BindingAgents

Purified or partially purified anti-GBV-C antibodies or binding agentsmay be administered to a subject for prophylatic or therapeutictreatment of HIV. Passive immunization has been administered for severalbacterial infections including pneumococcal pneumonia and H. influenzapneumonia. In pneumococcal disease it was essential to identify theinfecting serotype and obtain the appropriate type specific antiserum.The problems that arose from using horse serum and the difficulty inprecisely defining the serotype led to the abandonment of this procedureas soon as antibiotic therapy was introduced into clinical medicine.

In recent years, passive immunotherapy has been used for several viraldiseases such as hepatitis A, hepatitis B, polio, etc., and the use ofintravenous X-globulin has grown as its applications have expanded.There have been several clinical trials with human monoclonal antibodiesin various infectious diseases that document not only efficacy but alsosafety. It is contemplated therefore, that antibodies to GBV-C derivedepitopes that have similar structural attributes to an infectivepathogen, such as HIV, may be effective in either preventing infectionsor in actual therapy.

Although the bulk of contemporary opinion in virology and immunologysupports the prevailing paradigm that immunity to the humanimmunodeficiency virus is largely cellular in nature, a significant bodyof evidence in vaccine studies in animals suggest a pivotal role for thehumoral immune system (Sawyer et al., 1990, Moore et al., 1991). Inchronic viral infections, antibodies may be critical at certain stages.As such, antibodies may play a crucial role in the control of HIV-1infections. In particular, through the use of the present invention HIVmay be inhibited in its ability to infect the body, or at least thereduce the level of infection or replication.

B. Active Immunotherapy Related to GBV-C Antigens

Certain embodiments of the invention include the vaccination of asubject with an antigen derived from a GBV-C protein, in particular aGBV-C envelope protein, for the therapeutic or prophylactic treatment ofHIV. In certain embodiments, the antigen can be all or part of the GBV-CE2 polypeptide or mimcs thereof. Appropriate mimetics may be designedbase on secondary or tertiary structure of a protein or peptide. Thisvaccination elicits the production of antibodies, i.e. GBV-C and HIVbinding agents.

In particular aspects, anti-HIV properties may be illicited byexpression or over-expression of a GBV-C antigen by an attenuated GBV-Cviral vector. Anti-HIV properties will typically results in themodification of an HIV infection or the sensitivity to such aninfection. Anti-HIV properties include, but are not limited to, delayingor slowing propagation of HIV; reducing viral load; reducing viralspread; reducing or limiting the severity of secondary pathologies, suchas opportunistic infections and the like; preventing or reducing theprobability of infection; neutralizing HIV particles; or competing withHIV binding sites on cells and in tissues and organs of a person exposedto HIV.

Active immunotherapy involves immunization of a subject to enhanceexisting or to elicit novel pathogen-specific immune responses, i.e., anHIV immune response, and, for example, provide systemic anti-pathogenimmunity. Immunotherapeutic vaccination is the concept of inducing orenhancing immune responses of the subject to antigenic determinants thatare uniquely expressed or expressed at increased levels on pathogens orcells infected by pathogens. Antigenic determinants may be in the formof peptides, polypeptides, attenuated pathogens, and the like.

The immune response is the way the body defends itself againstmicroorganisms, viruses, and other potentially harmful substances ororganisms. Antigens are typically molecules (usually proteins) on thesurface of cells, viruses, fungi, bacteria, and some non-livingsubstances such as toxins, chemicals, drugs, and foreign particles. Theimmune system recognizes and destroys substances containing theseantigens.

The immune response may be an active immune response. Active immunitydevelops when the body is exposed to various antigens (antigenicepitopes), such as those described herein. It involves lymphocytes, ofwhich there are 2 main groups, B-lymphocytes, and T lymphocytes. Blymphocytes (also called B cells) produce antibodies. Antibodies attachto a specific antigen and make it easier for the phagocytes to destroythe antigen. T lymphocytes (T cells) attack antigens directly, and someT lymphocytes provide control of the immune response. B cells and Tcells develop that are specific for an antigen type. When a subject isexposed to a different antigen, different B cells and T cells areformed.

1. B Cells

B cells are a type of lymphocyte. The B cell produces antibodies thatbind antigens. Each B cell is programmed to make a specific antibody.When a B cell encounters its antigen (along with collaborating T cellsand accessory cells), it gives rise to many large plasma cells. Everyplasma cell is a factory for producing antibody. Each of the plasmacells descended from a given B cell (which are all members of the samefamily, or clone) manufactures millions of identical antibody moleculesand pours them into the bloodstream.

A given antibody has an affinity for a particular antigen. Theantibody-antigen interaction marks the antigen or the cell displayingthe antigen for destruction. After the human body has recovered from adisease, B-cells produce memory cells that attack the disease-causingorganism if it invades again. This second response is much quicker thanthe first, thus preventing symptoms of the disease from occurring. Thesecond phase involves the formation of the memory B-cell pool andseeding of long-lived plasma cells to the bone marrow. Plasma cells areterminally differentiated and do not give rise to memory cells.

Development of memory T cells (CD4 and CD8) may occur after activation,cells differentiate into effector T cells. Memory T cells may begenerated from effector T cells. There may be two subsets of memorycells: quiescent, central memory cells that recirculate from blood tosecondary lymphoid organs, and effector memory cells that migratethrough tissues and deliver a very rapid response on reactivation withantigen.

2. Cytotoxic T Lymphocytes

In certain embodiments, T-lymphocytes are activated by contact with anantigen-presenting cell that is in contact with an antigen of theinvention.

T cells express a unique antigen binding receptor on their membrane(T-cell receptor), which can only recognize antigen in association withmajor histocompatibility complex (MHC) molecules on the surface of othercells. There are several populations of T cells, such as T helper cellsand T cytotoxic cells. T helper cells and T cytotoxic cells areprimarily distinguished by their display of the membrane boundglycoproteins CD4 and CD8, respectively. T helper cells secrete variouslymphokines that are crucial for the activation of B cells, T cytotoxiccells, macrophages, and other cells of the immune system. In contrast, aT cytotoxic cell that recognizes an antigen-MHC complex proliferates anddifferentiates into an effector cell called a cytotoxic T lymphocyte(CTL). CTLs eliminate cells of the body displaying antigen, such asvirus-infected cells and tumor cells, by producing substances thatresult in cell lysis.

CTL activity may be assessed in freshly isolated peripheral bloodmononuclear cells (PBMC), in a phytohaemaglutinin-stimulated IL-2expanded cell line established from PBMC (Bernard et al., 1998) or by Tcells isolated from a previously immunized subject and restimulated for6 days with dendritic cells infected with an adenovirus vectorcontaining antigen using standard 4 hr ⁵¹Cr release microtoxicityassays. One type of assay uses cloned T-cells. Cloned T-cells have beentested for their ability to mediate both perforin and Fasligand-dependent killing in redirected cytotoxicity assays (Simpson etal., 1998). The cloned cytotoxic T lymphocytes displayed both Fas- andperforin-dependent killing. An in vitro dehydrogenase release assay hasbeen developed that takes advantage of a fluorescent amplificationsystem (Page et al., 1998). This approach is sensitive, rapid, andreproducible and may be used advantageously for mixed lymphocytereaction (MLR). It may easily be further automated for large scalecytotoxicity testing using cell membrane integrity, and is thusconsidered in the present invention. In another fluorometric assaydeveloped for detecting cell-mediated cytotoxicity, the fluorophore usedis the non-toxic molecule alamarBlue (Nociari et al., 1998). ThealamarBlue is fluorescently quenched (i.e., low quantum yield) untilmitochondrial reduction occurs, which then results in a dramaticincrease in the alamarBlue fluorescence intensity (i.e., increase in thequantum yield). This assay is reported to be extremely sensitive,specific and requires a significantly lower number of effector cellsthan the standard ⁵¹Cr release assay.

In certain aspects, T helper cell responses can be measured by in vitroor in vivo assay with peptides, polypeptides, or proteins. In vitroassays include measurement of a specific cytokine release by enzyme,radioisotope, chromaphore, or fluorescent assays. In vivo assays includedelayed type hypersensitivity responses called skin tests, as would beknown to one of ordinary skill in the art.

3. Antigen Presenting Cells

In general, the term “antigen presenting cell” can be any cell thataccomplishes the goal of the invention by aiding the enhancement of animmune response (e.g., from the T-cell or -B-cell arms of the immunesystem) against an antigenic composition of the present invention or aheterologous antigen or a immunologically functional equivalent. Suchcells can be defined by those of skill in the art, using methodsdisclosed herein and in the art. As is understood by one of ordinaryskill in the art (see for example Kuby, 1993, incorporated herein byreference), and used in certain embodiments, a cell that displays orpresents an antigen normally or preferentially with a class II majorhistocompatibility molecule or complex to an immune cell is an “antigenpresenting cell.” In certain aspects, a cell (e.g., an APC cell) may befused with another cell, such as a recombinant cell or a tumor cell thatexpresses the desired antigen. Methods for preparing a fusion of two ormore cells is well known in the art, such as for example, the methodsdisclosed in Goding, pp. 65-66, 71-74, 1986; Campbell, pp. 75-83, 1984;Kohler and Milstein (1975); Kohler and Milstein (1976), Gefter et al.(1977), each incorporated herein by reference. In some cases, the immunecell to which an antigen-presenting cell displays or presents an antigento is a CD4+ TH cell. Additional molecules expressed on the APC or otherimmune cells may aid or improve the enhancement of an immune response.Secreted or soluble molecules, such as for example, cytokines andadjuvants, may also aid or enhance the immune response against anantigen. Such molecules are well known to one of skill in the art, andvarious examples are described herein.

The dendritic cell (DC) is the cell type best suited for vaccine antigendelivery, as they are the most potent antigen presenting cells,effective in the stimulation of both primary and secondary immuneresponses (Steinman, 1999; Celluzzi and Falo, 1997). It is contemplatedin the present invention that the exposure of dendritic cells with aGBV-C vaccine of the invention, will elicit a potent immune responsespecific for the vaccine or vaccine vector of the present invention. Amore detailed description of vaccines is provided below.

C. Adoptive Immunotherapy Related to GBV-C Antigens

In various embodiments of the invention, it is contemplated that theantigens or anti-idiotypic antibodies may be used to stimulateautologous or heterologous immunocompetent cells for the treatment ofHIV. Adoptive immunotherapy is a technique that involves either removingimmunocompetent cells from the body, artificially increasing the number,and returning them to the body; or artificially altering target cells tomake them more immunogenic.

Typical adoptive immunotherapy involves the administration ofimmunologically active cells to an individual for providing a beneficialimmunological effect such as reduction or control of viral infections.The immunologically active cells are typically taken by venipuncture orleukophoreses either from the individual to be treated, termedautologous treatment, or from another individual, termed an allogeneicor heterologous treatment. The lymphocytes are then cultured to increasetheir number and to activate their therapeutic activity, and theninfused back into the patient. Thus, the majority of conventionalefforts in adoptive immunotherapy are typically directed at expandingcell numbers in vitro followed by infusion back into the patient.

Immunocompetent cells that may be used in adoptive immunotherapy are Tlymphocytes. A method for the activation of T lymphocytes to generateT-activated killer cells (T-AK) has been described as taking lymphocytesby leukophoresis or from peripheral blood, and stimulating said cellswith a monoclonal antibody (MAb) to a T cell surface receptor such asanti-CD3 (soluble or solid phase bound). The T cells can be stimulatedwith or without the addition of one or more cytokines such as IL-2.Alternatively, T cells can be purified before stimulation with the MAbto a surface receptor. Experimentation with T-AK cells has demonstratedthat CD8+ cells are responsible for the non-MHC restricted cytolyticactivity seen in these cultures (Anderson et al., 1989; Loeffler et al.,1991). The ability of IL-2 to expand T lymphocytes having immunereactivity and the ability to lyse fresh autologous, syngeneic, orallogeneic natural killer (NK) cell-resistant tumor cells, but notnormal cells, has resulted in the development of cell transfertherapies, such as autologous adoptive immunotherapy. Immunocompetentcells may include T lympocytes, dendritic cells, and the like.

V. Anti-GBV-C Antibodies or Binding Agents

Embodiments of the invention may include polypeptides in the form ofantibodies, single chain antibodies and the like that bind various GBV-Cpolypeptides, peptides, or derivatives thereof. Means for preparing andcharacterizing antibodies are well known in the art (see, e.g., Harlowand Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988; and Humphreys and Glover, 2001, each of which isincorporated herein by reference).

A. Anti-GBV-C Antibody Generation

The present invention provides therapeutic uses for anti-GBV-Cantibodies. In some embodiments, monoclonal antibodies as well aspolyclonal antibodies against GBV-C antigens may be used effectively inpreventive and therapeutic treatment of HIV. Thus, the present inventionis directed to anti-GBV-C antibody/antibodies that bind a GBV-C protein,polypeptide, or peptide, and attenuate HIV virus infectivity orreplication. In particular, antibodies that bind a GBV-C envelopeprotein, polypeptide, or peptide are contemplated. In particularembodiments, antibodies that bind a GBV-C E2 protein, polypeptide, orpeptide, as described herein, are contemplated. The invention alsocontemplates the use of a biologically functional equivalent of ananti-GBV-C antibody or a GBV-C antigen. The term “GBV-Cprotein/peptide/polypeptide” or “GBV-C antigen” is used herein to referto a GBV-C protein, polypeptide or peptide, irrespective of whether itoccurs naturally, is purified, is partially purified, or is produced byrecombinant DNA methods, fusion-protein methods, protein synthesismethods or is a biological functional equivalent thereof.

A biologically functional equivalent is molecule where modificationsand/or changes may be made in the structure of the polynucleotidesencoding and/or the protein molecule, while obtaining molecules havingsimilar or improved characteristics. In context of this invention, themolecule may be either a GBV-C antigen or an anti-GBV-C antibody. Thebiological functional equivalent may comprise a polynucleotide that hasbeen engineered to contain distinct sequences while at the same timeretaining the capacity to encode a “wild-type” or a functionalpolypeptide or peptide. This can be accomplished through the degeneracyof the genetic code, i.e., the presence of multiple codons, which encodefor the same amino acids. Methods for preparing such equivalents arewell known in the art.

The term “antibody” is used to refer to any antibody-like molecule thathas an antigen binding region, and includes antibody fragments such asFab′, Fab, F(ab′)₂, single domain antibodies (DABs), Fv, scFv (singlechain Fv or single chain antibodies), chimeras and the like. Methods andtechniques of producing the above antibody-based constructs andfragments are well known in the art (U.S. Pat. Nos. 5,889,157;5,821,333; 5,888,773, each specifically incorporated herein byreference).

1. Polyclonal Antibodies

A polyclonal antibody typically is prepared by immunizing an animal withan immunogenic composition (comprising a GBV-C antigen, for example) andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically, theanimal used for production of anti-antisera is a rabbit, a mouse, a rat,a hamster, a guinea pig, or a goat. Because of the relatively largeblood volume of rabbits, a rabbit is a preferred choice for productionof polyclonal antibodies.

As well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary carriers are keyhole limpet hemocyanin(KLH) and bovine serum albumin (BSA). Other proteins such as ovalbumin,mouse serum albumin, rabbit serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor can also be used as carriers. Means forconjugating a polypeptide to a carrier protein are also well known inthe art. Exemplary methods of conjugation include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimyde, andbis-biazotized benzidine. Other bifunctional or derivatizing agent mayalso be used for linking, for example maleimidobenzoyl sulfosuccinimideester (conjugation through cysteine residues), N-hydroxysuccinimide(through lysine residues), glutaraldehyde, succinic anhydride, SOCl₂, orR¹N═C═NR, where R and R¹ are different alkyl groups.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous, andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization.

A second, booster injection, also may be given. The process of boostingand titering is repeated until a suitable titer is achieved. When adesired level of immunogenicity is obtained, the immunized animal can bebled and the serum isolated and stored, and/or the animal can be used togenerate mAbs.

2. Monoclonal Antibodies

A “monoclonal antibody” refers to homogenous populations ofimmunoglobulins that are capable of specifically binding to a GBV-Cprotein. It is understood that the GBV-C protein or peptide, asdescribed herein, may have one or more antigenic determinants. Theantibodies of the invention may be directed against one or more of thesedeterminants.

Monoclonal antibodies (mAbs) may be readily prepared through use ofwell-known techniques, such as those exemplified in U.S. Pat. No.4,196,265, incorporated herein by reference. Typically, this techniqueinvolves immunizing a suitable animal with a selected immunogencomposition, e.g., a purified or partially purified GBV-C antigenprotein, polypeptide, or peptide. The immunizing composition isadministered in a manner effective to stimulate antibody-producingcells.

The methods for generating mAbs generally begin along the same lines asthose for preparing polyclonal antibodies. Rodents such as mice and ratsare preferred animals, however, the use of rabbit, sheep, goat, monkeycells is also possible. The use of rats may provide certain advantages(Goding, 1986, pp. 60-61), but mice are preferred, with the BALB/c mousebeing most preferred. The BALB/c mouse is most routinely used andgenerally gives a higher percentage of stable fusions.

The animals are injected with antigen, generally as described above.Following immunization, somatic cells with the potential for producingantibodies, specifically B-lymphocytes (B-cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens or lymph nodes. Spleen cells and lymph node cells arepreferred, the former because they are a rich source of antibodyproducing cells that are in the dividing plasmablast stage.

Often, a panel of animals will have been immunized and the spleen ofanimals with the highest antibody titer will be removed. The spleenlymphocytes are obtained by homogenizing the spleen with a syringe.

The antibody-producing B-lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984;each incorporated herein by reference). For example, where the immunizedanimal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1,Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bu1; forrats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266,GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection withhuman cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant-cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 proportion, though the proportion may vary fromabout 20:1 to about 1:1, respectively, in the presence of an agent oragents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKohler and Milstein (1975; 1976), and those using polyethylene glycol(PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use ofelectrically induced fusion methods also is appropriate (Goding pp.71-74, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, infusedcells (particularly the infused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides(hypoxanthine-aminopterin-thymidine (HAT) medium). Where azaserine isused, the media is supplemented with hypoxanthine. One preferredselection medium is HAT. Only cells capable of operating nucleotidesalvage pathways are able to survive in HAT medium.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple, and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways.

A sample of the hybridoma can be injected (often into the peritonealcavity) into a histocompatible animal of the type that was used toprovide the somatic and myeloma cells for the original fusion (e.g., asyngeneic mouse). Optionally, the animals are primed with a hydrocarbon,especially oils such as pristane (tetramethylpentadecane) prior toinjection. The injected animal develops tumors secreting the specificmAb produced by the fused cell hybrid. The body fluids of the animal,such as serum or ascites fluid, can then be tapped to provide mAbs inhigh concentration.

The individual cell lines could also be cultured in vitro, where themAbs are naturally secreted into the culture medium from which they canbe readily obtained in high concentrations.

mAbs produced by either means may be further purified, if desired, usingfiltration, centrifugation, and various chromatographic methods such asFPLC or affinity chromatography. Fragments of the mAbs of the inventioncan be obtained from the purified mAbs by methods that include digestionwith enzymes, such as pepsin or papain, and/or by cleavage of disulfidebonds by chemical reduction. Alternatively, mAb fragments encompassed bythe present invention can be synthesized using an automated peptidesynthesizer.

It also is contemplated that a molecular cloning approach may be used togenerate monoclonals. For this, combinatorial immunoglobulin phagemidlibraries are prepared from RNA isolated from the spleen of theimmunized animal, or prom human cells derived from non-immunizedindividuals, and phagemids expressing appropriate antibodies areselected by panning using cells expressing the antigen and controlcells. The advantages of this approach over conventional hybridomatechniques are that approximately 10⁴ times as many antibodies can beproduced and screened in a single round, and that new specificities aregenerated by H and L chain combination which further increases thechance of finding appropriate antibodies. A second advantage ofmonoclonal antibody production by screening recombinant libraries is thelack of need for immunization and a third is the ability to producetotally human monoclonal antibodies. Two commercially availableanti-GBV-C E2 monoclonal antibodies have been tested for HIV-inhibitoryeffects on an R5 HIV strain. The M6 (Roche) was the best, but all threeinhibited HIV. This was done in duplicate, and is similar to resultsseen in another previous experiment. The Roche monoclonal antibody hasbeen studied for binding to E2 protein by pepscan. The antibodyrecognizes a linear epitope found on two overlapping peptidesrepresenting the GBV-C E2 protein. Thus, the epitope may be contained ina 9 amino acid sequence FYEPLVRRC (SEQ ID NO:8). This amino acid may bean HIV disease-modifying immunogen (Vaccine). The peptide may be used toinhibit HIV (as a prototype drug), and induce anti-HIV antibodies (as avaccine).

Antibodies from Roche are described in Tacke et al. (1997) and Schmolkeet al. (1998). For a description of epitope mapping studies see Schmolkeet al. (1998). A “BD” and “VS” antibodies are commercially availablemonoclonal antibodies against GBV-C E2 sold by Biodesign and Virostat,respectively. Other Roche antibodies may also demonstrate these sameaffects, M113, and M30.

3. Humanized Anti-GBV-C Antibodies

In certain embodiments of the invention, anti-GBV-C antibodies may behumanized for therapeutic purposes. Humanized mAbs are antibodies ofanimal origin that have been modified using genetic engineeringtechniques to replace constant regions and/or variable region frameworksequences with human sequences, while retaining the original antigenspecificity. Such antibodies can also include a humanized heavy chainassociated with a donor or acceptor unmodified light chain or a chimericlight chain, or vice versa. Such antibodies are commonly derived fromrodent antibodies, for example, the murine Ab of the present invention.Rodent derived antibodies may demonstrate a specificity against humanantigens and are generally useful for in vivo therapeutic applications.This strategy reduces the host response to the foreign antibody andallows selection of the human effector functions.

The techniques for producing humanized immunoglobulins are well known tothose of skill in the art. For example U.S. Pat. No. 5,693,762 disclosesmethods for producing, and compositions of, humanized immunoglobulinshaving one or more complementarity determining regions (CDR's). “CDRs”are defined as the complementarity determining region amino acidsequences of an antibody. CDRs are contained within the hypervariableregions of immunoglobulin heavy and light chains. CDRs provide themajority of contact residues for the binding of the antibody to theantigen or epitope. CDRs of interest in this invention are derived fromdonor antibody variable heavy and light chain sequences, and includefunctional fragments and analogs of the naturally occurring CDRs, whichfragments and analogs also share or retain the same antigen bindingspecificity and/or neutralizing ability as the donor antibody from whichthey were derived. When combined into an intact antibody, the humanizedimmunoglobulins are substantially non-immunogenic in humans and retainsubstantially the same affinity as the donor immunoglobulin to theantigen, such as a protein or other compound containing an epitope.

Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source that is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. A humanizedantibody is one in which only the antigen-recognized sites, orcomplementarity-determining hypervariable regions (CDRs) are ofnon-human origin, whereas all framework regions (FR) of variable domainsare products of human genes.

Humanization can be essentially performed following the method of Winterand co-workers (Jones et al., 1986; Riechmann et al., 1988; Verhoeyen etal., 1988), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies, wherein less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some CDR residues and possibly someframework region (FR) residues are substituted by residues fromanalogous sites in rodent antibodies.

Other U.S. Patents, each incorporated herein by reference, that teachthe production of antibodies useful in the present invention includeU.S. Pat. No. 5,565,332, which describes the production of chimericantibodies using a combinatorial approach; U.S. Pat. No. 4,816,567 whichdescribes recombinant immunoglobin preparations and U.S. Pat. No.4,867,973 which describes antibody-therapeutic agent conjugates.

U.S. Pat. No. 5,565,332, which incorporated herein by reference,describes methods for the production of antibodies, or antibodyfragments, which have the same binding specificity as a parent antibody,but have increased human characteristics. Humanized antibodies may beobtained by chain shuffling, perhaps using phage display technology.Human antibodies may also be produced by transforming B-cells with EBVand subsequent cloning of secretors as described by Hoon et al., (1993).

4. Human Anti-GBV-C Antibodies

Embodiments of the invention may use human monoclonal antibodies incompositions and methods described herein. Human mAbs can be made usinga hybridoma method. Human myeloma and mouse-human heteromyeloma celllines for the production of human mAbs have been described, for example,by Kozbor (1984), and Brodeur et al. (1987).

It is now possible to produce transgenic animals (e.g., mice) that arecapable, upon immunization, of producing a repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge (Jakobovits et al., 1993).

Alternatively, phage display technology can be used to produce humanantibodies and antibody fragments in vitro, from immunoglobulin variable(V) domain gene repertoires from unimmunized donors (McCafferty et al.,1990). According to this technique, antibody V domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, such as M13 or fd, and displayed as functional antibodyfragments on the surface of the phage particle.

Because the filamentous particle contains a single-stranded DNA copy ofthe phage genome, selections based on the functional properties of theantibody also result in selection of the gene encoding the antibodyexhibiting those properties. Thus, the phage mimics some of theproperties of the B-cell. Phage display can be performed in a variety offormats (Johnson et al., 1993). Several sources of V-gene segments canbe used for phage display. A repertoire of V genes from unimmunizedhuman donors can be constructed and antibodies to a diverse array ofantigens (including self-antigens) can be isolated essentially followingthe techniques described by Marks et al. (1991), or Griffith et al.(1993).

In a natural immune response, antibody genes accumulate mutations at ahigh rate (somatic hypermutation). Some of the changes introduced willconfer higher affinity, and B-cells displaying high-affinity surfaceimmunoglobulin are preferentially replicated and differentiated duringsubsequent antigen challenge. This natural process can be mimicked byemploying the technique known as “chain shuffling” (Marks et al., 1992).In this method, the affinity of “primary” human antibodies obtained byphage display can be improved by sequentially replacing the heavy andlight chain V region genes with repertoires of naturally occurringvariants (repertoires) of V domain genes obtained from unimmunizeddonors. This techniques allows the production of antibodies and antibodyfragments with affinities in the nM range. A strategy for making verylarge phage antibody repertoires has been described by Waterhouse et al.(1993), and the isolation of a high affinity human antibody directlyfrom such large phage library is reported by Griffith et al. (1993).

5. Anti-GBV-C Antibody Conjugates

Antibody conjugates comprising a GBV-C antibody linked to another agent,such as but not limited to a therapeutic agent, a anti-viral agent, adetectable label, a cytotoxic agent, a chemical, a toxic, an enzymeinhibitor, a pharmaceutical agent, etc. form further aspects of theinvention. Antibody conjugates may be used both in in vitro diagnosticsand in a variety of immunoassays.

Certain antibody conjugates include may be for use in vitro, where theantibody is linked to a secondary binding ligand or to an enzyme (anenzyme tag) that will generate a colored product upon contact with achromogenic substrate. Examples of suitable enzymes include urease,alkaline phosphatase, (horseradish) hydrogen peroxidase and glucoseoxidase. Preferred secondary binding ligands are biotin and avidin orstreptavidin compounds. The use of such labels is well known to those ofskill in the art and is described, for example, in U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and4,366,241, each incorporated herein by reference.

6. Single Chain Antibodies

The Fv portion of an antibody is a 26 kDa heterodimer consisting of theamino-terminal variable domains of the heavy and light chains, and isthe smallest fragment to bear the antigen binding site. Geneticallyengineered single chain Fv (Fv) peptides have been synthesized byattaching the carboxyl terminus of one variable domain to the aminoterminus of the other with a peptide linker. These Fv fragments havebeen shown to bind specific antigens, like the transferrin receptor,have been used to localize fusion proteins to targeted cells.

VI. Anti-HIV GBV-C Vaccines

The present invention includes methods for preventing the development ofor treating AIDS in both infected and uninfected persons, as well as theelicitation or enhancement of an immune response. As such, the inventioncontemplates vaccines for use in active, passive, and adoptiveimmunization embodiments. Immunogenic compositions, proposed to besuitable for use as active vaccines, may be prepared from an infectious,conditionally replicative, or replication defective GBV-C nucleic acid.Immunogenic compositions may also be prepared from a recombinantexpression construct or synthesized in a manner disclosed herein or isknown in the art. Preferably the antigenic material is extensivelydialyzed to remove undesired small molecular weight molecules and/orlyophilized for more ready formulation into a desired vehicle.

The present invention, in certain embodiments, involves the use of aGBV-C antigen, for example, and E2 antigen. The antigen may also be afragment of a GBV-C virus protein, such as a peptide (discussed above).In a particular embodiment, the antigen is contemplated that containsthe 9 amino acid sequence FYEPLVRRC (SEQ ID NO:8). In preferredembodiments, the antigen is contemplated to comprise the 13 amino acidLTGGFYEPLVRRC (SEQ ID NO:6). In more preferred embodiments, the antigenis contemplated to comprise the 17 amino acid sequence GGAGLTGGFYEPLVRRC(SEQ ID NO:7). This amino acid may be an HIV disease-modifying immunogen(vaccine) and/or induce anti-HIV antibodies (as a vaccine).

A. Carrier Molecules for Vaccination Against GBV-C Antigens

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling the heterologous polypeptideimmunogen to a carrier. Exemplary carriers are keyhole limpet hemocyanin(KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin,mouse serum albumin, or rabbit serum albumin can also be used ascarriers. Means for conjugating a polypeptide to a carrier protein arewell known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimyde, andbis-biazotized benzidine.

B. Adjuvants

As is also well known in the art, the immunogenicity of a polypeptide orpeptide composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Suitableadjuvants include all acceptable immunostimulatory compounds, such ascytokines, toxins, or synthetic compositions.

Adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, IL-12,γ-interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such asthur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A(MPL). RIBI, which contains three components extracted from bacteria,MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2%squalene/Tween 80 emulsion. MHC antigens may even be used.

Exemplary, often preferred adjuvants include complete Freund's adjuvant(a non-specific stimulator of the immune response containing killedMycobacterium tuberculosis), incomplete Freund's adjuvants and aluminumhydroxide adjuvant.

In addition to adjuvants, it may be desirable to coadminister biologicresponse modifiers (BRM), which have been shown to upregulate T cellimmunity or downregulate suppresser cell activity. Such BRMs include,but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA);or low-dose Cyclophosphamide (CYP; 300 mg/m²) (Johnson/Mead, NJ) andcytokines such as γ-interferon, IL-2, or IL-12 or genes encodingproteins involved in immune helper functions, such as B-7.

VII. Anti-HIV Therapies

In certain embodiments, therapeutic methods will include administeringto a patient or subject a composition comprising an antigen or anantibody derived from a GBV-C polypeptide. In various embodiments, thetreatment methods of the invention may be used in combination with otheranti-HIV treatments, such as GBV-C infection as a therapeutic orpreventative treatment for AIDS. For exemplary compositions and methodssee PCT application WO 01/77157, which is incorporated herein byreference.

As a therapeutic measure, a binding agent that binds a GBV-C derivedamino acid molecule can be used to reduce the severity or progression ofAIDS, including the prevention of AIDS in HIV-infected individuals. Areduction in severity or progression of AIDS includes, but is notlimited to, prevention of or a reduction in the severity, duration, ordiscomfort associated with the following conditions: prolonged andunexplained fatigue; swollen glands; prolonged fever; chills; excessivesweating; swollen gums and mouth lesions; sore throat; cough; shortnessof breath; constipation; diarrhea; symptoms of well-known opportunisticinfections; Kaposi sarcomas; skin rashes or lesions; loss of appetite orweight loss; malaise; headaches; speech impairment; muscle atrophy;memory loss; reduced cognitive functioning; swelling of the joints;joint stiffness or pain; cold intolerance; pain or tenderness in bones;energy level; anxiety, stress, and tension; groin lump; pruritus;genital sores; blurred or decreased vision; diplopia; light sensitivity;pain in chest, sides, back, muscle or stomach; and seizures.

As a preventative measure, a patient may be administered apharmaceutically acceptable composition comprising a HIV neutralizing orattenuating binding agent derived from a GBV-C polypeptide. The anti-HIVGBV-C binding agent may be used in conjunction with infection of CD4+ Tcells with GBV-C or a recombinant version of GBV-C to inhibit infectionof these cells by HIV. Alternatively, treatment with the GBV-Ccompositions of the present invention may effect a combination ofpreventative and therapeutic treatments insofar as infection of othercells in an HIV-infected subject's body is prevented or attenuated.

Inhibition of AIDS progression may be demonstrated by reduction ofdetectable HIV in the HIV-infected subject; maintaining a CD4 countabove 200 for a longer than average period of time; maintaining a normalT cell count; or maintaining normal p24 antigen. The term “therapeuticbenefit” or “therapeutic effect” used throughout this application refersto anything that promotes or enhances the well-being of the subject withrespect to the medical treatment of his/her condition, which includestreatment of HIV-infection (before the onset of AIDS), AIDS, as well astreatment of Hepatitis C. A list of nonexhaustive examples of thisincludes extension of the subject's life by any period of time; decreaseor delay in the progression of AIDS (HIV, as described above) orHepatitis C; decrease in viral load of HIV or HCV; decrease in HIVreplication; clearance of HIV or HCV viremia reduced transmission of HCVor HIV; decrease in liver damage or complications; and a decrease inpain to the subject that can be attributed to the subject's condition.

A. Combination Therapies

Of course it is understood that the method of the present invention,particularly administration of agents that bind a GBV-C amino acidmolecule as treatment for an HIV-infected subject, may also be used incombination with the administration of traditional therapies.Alternatively, the compositions of the present invention may be given incombination with treatment or prevention of hepatitis C, such asα-interferon. Some such therapies are described below.

In many clinical situations, it is advisable to use a combination ofdistinct therapies. Thus, it is envisioned that, in addition to thetherapies described herein, one would also wish to provide to thepatient more “standard” pharmaceutical anti-retroviral therapies.Examples of standard therapies are provided below.

Combinations may be achieved by administering to a patient a singlecomposition or pharmacological formulation that includes both agents, orby administering to a patient two distinct compositions or formulations,at the same time, wherein one composition may include a GBV-C bindingagent, GBV-C antigen, or expression construct encoding a binding agentor antigen, and the other includes the standard anti-retroviral therapy.Alternatively, a GBV-C based therapeutic may precede or follow the othertreatment by intervals ranging from minutes to weeks. In embodimentswhere the other agent and GBV-C based therapeutic are adminsteredseparately to the patient, one would generally ensure that a significantperiod of time did not expire between the time of each delivery, suchthat the agent and GBV-C based therapeutic would still be able to exertan advantageously combined effect on the patient. In such instances, itis contemplated that one would administer to the patient both modalitieswithin about 12-24 hours of each other and, more preferably, withinabout 6-12 hours of each other, with a delay time of only about 12 hoursbeing most preferred. In some situations, it may be desirable to extendthe time period for treatment significantly, however, where several days(2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) lapsebetween the respective administrations.

It also is conceivable that more than one administration of a GBV-Cbased therapeutic agent will be desired. Various combinations may beemployed, where a GBV-C based therapeutic is “A” and the other agent is“B,” as exemplified below:A/B/A  B/A/B  B/B/A  A/A/B  B/A/A  A/B/B  B/B/B/A B/B/A/B A/A/B/BA/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/AA/A/B/A A/B/B/B B/A/B/B B/B/A/B

Other combinations are contemplated as well.

1. AZT

A well known, traditional therapy for the treatment of AIDS involveszovidovudine (AZT™ available from Burroughs Wellcome). This is one of aclass of nucleoside analogues known as dideoxynucleosides which blockHIV replication by inhibiting HIV reverse transcriptase. The anti-AIDSdrug zidovudine (also known as AZT) may also be used in limitedcircumstances, mostly in combination with rifampin, as described byBurger et al. (1993).

The compositions and methods disclosed herein will be particularlyeffective in conjunction with other forms of therapy, such as AZT and/orprotease inhibitors that are designed to inhibit viral replication, bymaintaining desirable levels of white blood cells. This, in effect, buysthe patient the time necessary for the anti-viral therapies to work.

2. HAART

New combination drug therapy has shown promising results in thetreatment of HIV-infected patients. Treatment with potent anti-HIV drugcombinations is referred to as “highly active anti-retroviral therapy”(HAART), and it has provided clinical improvement, longer survival, andimproved quality of life for people infected with HIV during all fourstages of HIV disease. Examples of HAART include a protease inhibitor(indinavir, nelfinavir, ritonavir, ritonavir/saquinavir, or saquinavir)combined with two nucleoside analogs (AZT/ddI, d4T/ddI, AZT/ddC,AZT/3TC, or d4T/3TC).

In many instances, it will be desirable to have multiple administrationsof the inventive compositions and/or a vaccines, usually not exceedingsix administrations or vaccinations, more usually not exceeding fourvaccinations. In certain embodiments, one or more, usually at leastabout three administrations or vaccinations may be provided. Theadministrations or vaccinations will normally be at from two to twelveweek intervals, more usually from three to five week intervals. Periodicboosters at intervals of 1-5 years, usually three years, will bedesirable to maintain protective levels of the antibodies. The course ofthe immunization or treatment may be followed by standard antibodyassays. The assays may be performed by labeling with conventionallabels, such as radionuclides, enzymes, fluorescents, and the like.These techniques are well known and may be found in a wide variety ofpatents, such as U.S. Pat. Nos. 3,791,932; 4,174,384 and 3,949,064, asillustrative of these types of assays.

The manner of application may be varied widely. Any of the conventionalmethods for administration of an antibody or vaccine are applicable.These are believed to include oral application on a solidphysiologically acceptable base or in a physiologically acceptabledispersion, parenterally, by injection or the like. The dosage of theanti-GBV-C antibody or vaccine will depend on the route ofadministration and will vary according to the size of the host.

The anti-GBV-C binding agents, GBV-C infectious nucleic acids and/orGBV-C antigens of the invention may be formulated into apharmaceutically acceptable composition, see below, or vaccine asneutral or salt forms. Pharmaceutically-acceptable salts include theacid addition salts (formed with the free amino groups of the peptide)and those that are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups may also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

The preparation of binding agent that bind GBV-C sequences as activeingredients is generally well understood in the art by analogy, asexemplified by U.S. Pat. Nos. 6,479,243, 6,399,763, 5,714,153,5,582,981, and 4,833,077, all incorporated herein by reference. Thepreparation of vaccines that contain GBV-C sequences as activeingredients is generally well understood in the art by analogy, asexemplified by U.S. Pat. Nos. 5,958,895, 6,004,799, and 5,620,896, allincorporated herein by reference.

VIII. Pharmaceutical Compositions and Routes of Administration

Pharmaceutical compositions, including the immunoglobulins for passiveimmunotherapy or antigens for active immunotherapy, are typically usedfor prophylaxis of susceptible individuals and for the treatment ofinfections. A discussion of passive and active immunity and immunizingagents may be found in Remington's Pharmaceutical Sciences, 1990. Theimmunity provided by passive immunization is typically not long lastingand the immunoglobulins provided leave the body tissues and fluids ofthe host within a comparatively short period of time, usually after oneto two weeks, either by utilization by binding to the pathogen or bymetabolism by the host's body. Thus, the administration of an antibodyfor passive immunity may be during the critical period immediately afteror just prior to the predicted exposure to the pathogen or toxin suchthat the immunoglobulins are present when immunity is most urgentlyrequired.

The percentage of active compound in any pharmaceutical preparation isdependent upon both the activity of the compound, in this case bindingof an antibody(ies) or other binding agent, and its concentration in thepreparation. Typically, such compositions should contain at least 0.1%active compound. The percentage of the compositions and preparationsmay, of course, be varied and may conveniently be between about 2 toabout 60% of the weight of the unit. The amount of active compounds insuch therapeutically useful compositions is such that a suitable dosagewill be obtained.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy injection is possible. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal,phenylmecuric nitrate, m-cresol, and the like. In many cases, it will bepreferable to use isotonic solutions, for example, sugars or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate, and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed bysterile filtration. Generally, dispersions are prepared by incorporatingthe various sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying techniques that yield apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

The present invention contemplates GBV-C antigens, anti-GBV-Cantibodies, and/or infectious GBV-C nucleic acid molecules as well asinfectious nucleic acid molecules encoding, in some embodiments, aheterologous sequence, collectively “therapeutic GBV-C compositions”. Insome embodiments, pharmaceutical compositions are administered to asubject. Different aspects of the present invention involveadministering an effective amount of an aqueous composition. In anotherembodiment of the present invention, therapeutic GBV-C compositions areadministered to a subject to either prevent the infection by HIV orprevent the progression of HIV infection to development of AIDS.Additionally, such compounds can be administered in combination withtreatment by HAART or by administration of AZT and/or other anti-HIVdrugs or drug regiments. Though typically, anti-GBV-C agent or GBV-Cantigens will be administered separately from medication. Suchcompositions will generally be dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. Those of skill inthe art are well aware of how to apply antibodies or other bindingagents, as well as gene delivery to in vivo and ex vivo situations.

The phrases “pharmaceutically acceptable” or “pharmacologicallyacceptable” refer to molecular entities and compositions that do notproduce an adverse, allergic, or other untoward reaction whenadministered to an animal, or human, as appropriate. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifingal agents,isotonic and absorption delaying agents, and the like. The use of suchmedia and agents for pharmaceutical active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredients, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients, such asother anti-cancer agents, can also be incorporated into thecompositions.

In addition to the compounds formulated for parenteral administration,such as those for intravenous or intramuscular injection, otherpharmaceutically acceptable forms include, e.g., tablets or other solidsfor oral administration; time release capsules; and any other formcurrently used, including cremes, lotions, mouthwashes, inhalants andthe like.

The active compounds of the present invention can be formulated forparenteral administration, e.g., formulated for injection via theintravenous, intramuscular, intrathoracic, sub-cutaneous, or evenintraperitoneal routes. Administration by i.v. or i.m. are specificallycontemplated.

The preparation of an aqueous composition that contains a compound orcompounds that increase the expression of an MHC class I molecule willbe known to those of skill in the art in light of the presentdisclosure. Typically, such compositions can be prepared as injectables,either as liquid solutions or suspensions; solid forms suitable for useto prepare solutions or suspensions upon the addition of a liquid priorto injection can also be prepared; and, the preparations can also beemulsified.

The antibodies, binding agents, or other active compositions may beformulated as neutral or salt forms. Pharmaceutically acceptable salts,include the acid salts and those which are formed with inorganic acidssuch as, for example, hydrochloric or phosphoric acids, or such organicacids as acetic, oxalic, tartaric, mandelic, and the like. Salts formedwith the free carboxyl groups may also be derived from inorganic basessuch as, for example, sodium, potassium, ammonium, calcium, or ferrichydroxides, and such organic bases as isopropylamine, trimethylamine,2-ethylamino ethanol, histidine, procaine, and the like.

The course of the treatment may be followed by assays for antibodiesagainst antigens. The assays may be performed by labeling withconventional labels, such as radionuclides, enzymes, fluorescers, andthe like. Samples for assaying may be serum samples, or they may beobtained from any mucosal surface, or body fluid, such as saliva,sputum, vaginal wash, or expectoration. These assay techniques are wellknown and may be found in a wide variety of patents, such as U.S. Pat.Nos. 3,791,932; 4,174,384 and 3,949,064, as illustrative of these typesof assays.

In certain embodiments, it may be desirable to provide a continuoussupply of therapeutic compositions to the patient. For intravenous orintraarterial routes, this is accomplished by drip system. For variousapproaches, delayed release formulations could be used that providedlimited but constant amounts of the therapeutic agent over and extendedperiod of time. For internal application, continuous perfusion, forexample with an anti GBV-C antibody, binding agent, antigen and/or aGBV-C viral vector which may or may not carry a heterologous nucleicacid segment may be preferred. This could be accomplished bycatheterization followed by continuous administration of the therapeuticagent. The time period for perfusion would be selected by the clinicianfor the particular patient and situation, but times could range fromabout 1-2 hours, to 2-6 hours, to about 6-10 hours, to about 10-24hours, to about 1-2 days, to about 1-2 weeks or longer. Generally, thedose of the therapeutic composition via continuous perfusion will beequivalent to that given by single or multiple injections, adjusted forthe period of time over which the injections are administered. It isbelieved that higher doses may be achieved via perfusion, however.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 mL of isotonic NaCl solutionand either added to 1000 mL of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, Remington's PharmaceuticalSciences, 1990). Some variation in dosage will necessarily occurdepending on the condition of the subject being treated. The personresponsible for administration will, in any event, determine theappropriate dose for the individual subject.

An effective amount of the therapeutic composition is determined basedon the intended goal. The term “unit dose” or “dosage” refers tophysically discrete units suitable for use in a subject, each unitcontaining a predetermined-quantity of the therapeutic compositioncalculated to produce the desired responses, discussed above, inassociation with its administration, i.e., the appropriate route andtreatment regimen. The quantity to be administered, both according tonumber of treatments and unit dose, depends on the protection desired.

Antibodies or other binding agents may be administered in a dose thatcan vary from 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70,80, 90, 100 mg/kg of weight to 50, 60, 70, 80, 90, 100, 110, 120, 130,140, 150, 160, 170, 180, 190, 200 mg/kg of weight in one or more daily,weekly, monthly, or yearly administrations during one or various days,weeks, months, or years. The antibodies can be administered byparenteral injection (intravenous, intraperitoneal, intramuscular,subcutaneous, intracavity or transdermic). For viral vectors, onegenerally will prepare a viral vector stock. Depending on the kind ofvirus and the titer attainable, one will deliver 1 to 100, 10 to 50,100-1000, or up to 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰,1×10¹¹, or 1×10¹² infectious particles to the patient. Similar figuresmay be extrapolated for liposomal or other non-viral formulations bycomparing relative uptake efficiencies. Formulation as apharmaceutically acceptable composition is discussed below.

In many instances, it will be desirable to have multiple administrationsof the antibodies or other compositions of the invention. Thecompositions of the invention may be administered 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more times. The administrations will normally be at fromone to twelve week intervals, more usually from one to four weekintervals. Periodic re-administration will be desirable with recurrentexposure to the pathogen (e.g., HIV). For example, an HIV positivemother would be re-inoculated prior to parturition from a secondpregnancy.

Dosages commonly used for formulations that provide passive immunity arein the range of from 0.5 ml to 10 ml per dose, preferably in the rangeof 2 ml to 5 ml per dose. Repeated doses to deliver the appropriateamount of active compound are common. Both the age and size by weight ofthe recipient must be considered when determining the appropriate dosageof active ingredient and volume to administer.

Precise amounts of the therapeutic composition also depend on thejudgment of the practitioner and are peculiar to each individual.Factors affecting dose include physical and clinical state of thepatient, the route of administration, the intended goal of treatment(alleviation of symptoms versus cure) and the potency, stability, andtoxicity of the particular therapeutic substance.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,but drug release capsules and the like can also be employed.

As used herein, the term in vitro administration refers to manipulationsperformed on cells removed from an animal, including, but not limitedto, cells in culture. The term ex vivo administration refers to cellsthat have been manipulated in vitro, and are subsequently administeredto a living animal. The term in vivo administration includes allmanipulations performed on cells within an animal.

In certain aspects of the present invention, the compositions may beadministered either in vitro, ex vivo, or in vivo. In certain in vitroembodiments, transcribed RNA from a GBV-C clone is transfected into PBMCusing DEAE-dextran. The transduced cells can then be used for in vitroanalysis, or alternatively for in vivo administration.

U.S. Pat. Nos. 4,690,915 and 5,199,942, both incorporated herein byreference, disclose methods for ex vivo manipulation of bloodmononuclear cells and bone marrow cells for use in therapeuticapplications.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 HIV Neutralization Assay

E2 antibody serum was studied for interactions with HIV in a virusneutralization assay. In one study (FIG. 1), a clinical R5 strain of HIVwas incubated with a GBV-C RNA negative-E2 antibody negative serum(mock), with GBV-C E2 antibody positive-RNA negative serum (E2), GBV-CRNA positive-E2 negative serum (GBV-C), or a mixture of E2 and GBV-Csera for 1 hour at 37° C. prior to adding the mixture to PHA-IL-2stimulated peripheral blood mononuclear cells (PBMCs). After infection,cells were washed, and media was collected daily for 3 days for testingfor HIV p24 antigen (p24 Ag) in culture supernatant by ELISA. HIV p24 Agwas measured, and the percent inhibition determined by dividing theconcentration present in the test sample by the HIV-mock infectedcontrol sample. The baseline HIV p24 Ag was determined using the“mock”-HIV mixture, and significant inhibition of HIV replication (asmeasured by p24 Ag production into culture supernatant fluids) wasobserved when E2-positive sera was mixed with HIV, although thisinhibition declined each day. Similarly, when GBV-C RNA-positive sera(and GBV-C replication) was mixed with HIV, the HIV replication wasinhibited to a greater extent than with E2 antiserum alone, and themixture of E2 and GBV-C RNA positive sera gave the greatest extent ofHIV inhibition.

Reproducibility was determined by performing additional studies. Twoadditional GBV-C E2 antibody positive (RNA negative) sera (isolates 55and 9 respectively) and two E2 antibody negative (and RNA negative) sera(negative control sera 1 and 2) were studied for their ability todecrease replication of the R5 HIV strain. A clinical isolate was usedfor this study. In addition, diluted sera at 1:10, 1:100, and 1:1,000were used to determine if there was a dose-response relationship betweenthe concentration of serum and the extent of HIV inhibition. Afterwashing the PBMCs, sera was maintained in the culture media throughoutthe experiment, and infections were monitored on day 3 for HIV p24 Agproduction in culture supernatant. FIG. 2. Illustrates that HIV isinhibited by two E2 antibody positive sera in a dose-dependent fashion;whereas, E2 antibody negative sera do not inhibit HIV.

Example 2 HIV Neutralization Assay with Purified Antibody

To determine if the inhibitory substance in the GBV-C E2antibody-positive sera was antibody, IgG from four E2 antibody-positivesera and two E2 antibody-negative sera were purified by protein G columnchromatography. HIV was mixed with a “no antibody” control (No Ab), orwith 5 μg/ml of an E2 antibody negative control (NC-2) or E2 positivecontrols. The mixtures were applied to PBMCs, and after washing the HIVinocula, the cognate IgGs were maintained in the culture media. The rawp24 Ag results are shown in FIG. 3, and the percent HIV p24 Aginhibition is shown in FIG. 4.

Example 3 HIV Neutralization Assay on HIV Strain X4

Studies were performed to determine if E2 antibody-positive seruminhibited X4 strains of HIV. Using the same experimental design as usedfor FIGS. 3 and 4, IgG preparations from E2-negative and -positive IgGpreparations were studied for their ability to inhibit a clinical X4 HIVstrain in PHA-IL-2 PBMC cultures. Similar to the R5 strain, the X4strain was inhibited by GBV-C E2-positive IgG, but not GBV-C E2-negativeIgG (FIG. 5).

Since R5 viruses utilize CCR5 as their co-receptor, and X4 virusesutilize CXCR4 as their co-receptor, GBV-C E2 antibody inhibitionindicates that they are cross-reacting with a conserved epitope on HIVthat inhibits HIV replication, and that this epitope is on bothco-receptor usage types of HIV. Since the epidemiological data indicatesthat E2 antibody is associated with prolonged survival in Germany,France, and the United States, this interaction has promise for HIVstrains widely distributed worldwide.

Example 4 HIV Neutralization with Monoclonal Antibodies

Commercially available anti-GBV-C E2 monoclonal antibodies fromBiodesign and Virostat, and one suppplied by Roche were tested forHIV-inhibitory effects. Inhibition of an R5 HIV strain was detected whenusing all three antibodies, with M6 (Roche) being the best. Studies wereperformed in duplicate, and are similar to results seen in anotherprevious study. The Roche monoclonal antibody binding to E2 protein hasbeen studied using pepscan. M6 recognizes a linear epitope found on twooverlapping peptides representing the GBV-C E2 protein. Thus, an epitopeis contained in a 9 amino acid sequence of FYEPLVRRC (SEQ ID NO:8) or ina 17 amino acid sequence of GGAGLTGGFYEPLVRRC (SEQ ID NO:6). This aminoacid sequence may be an HIV disease-modifying immunogen (vaccine).

FIG. 6 demonstrates that the Roche M6 monoclonal antibody inhibits HIV-1(R5 strain). HIV was mixed with M6 antibody or an isotype controlantibody (range of concentrations, as shown) for 1 hr at 37° C., thenadded to PBMC cultures. Four hrs later, cells were washed, and media wasadded to cells (media containing either M6 or IC). Culture supernatantswere collected on day 3 post-infection and HIV p24 antigen determined.Percent inhibition was determined by dividing the p24 antigenconcentration in the M6 culture superantant by the Isotype control p24antigen concentration. This value was subtracted from 1, and the resultwas multiplied ×100.

FIG. 7 demonstrates that the biodesign and virostat antibodies alsoinhibited HIV-1, although not as efficiently as M6. FIG. 8 is apredicted map of the epitopes identified by the Roche mAbs as describedin Schmolke et al. (1998). M3 inhibits all of the groups of antibodies.M6 was the only antibody to react with a linear peptide in a PEPSCANanalysis. Antibodies against this M6 epitope were found to not appear tobe elicited during GBV-C infection.

Example 5 HIV Neutralization with Rabbit Sera

To determine if the peptide antigen shown to react with the anti-GBV-CE2 (M6) monoclonal antibody was antigenic and if it exhibited anti-HIVactivity, the inventors conjugated the 17 amino acid peptide to KLH) andimmunized 2 New Zealand White rabbits (commercially done by InVitrogen).IgG was purified from serum collected pre-immunization and at 8 weeks(following immunization and 2 boosts). Pre-immune and post-immuneanti-GBV-C E2 peptide rabbit IgG was incubated with R5 and X4 HIV for 1hour, and then added to primary PBMCs for 3 hours. Cells were thenwashed, and maintained in media containing either pre-immune orpost-immune IgG. HIV production into culture supernatant was measured byp24 antigen, and the post-immune IgG reproducibly reduced HIVinfectivity in both R5 and X4 viruses (FIGS. 10, 11, and 12). Allexperiments were performed in triplicate, and the reduction in p24antigen levels by post-immune IgG were all statistically significant atthe P<0.05 level. These studies demonstrate that anti-GBV-C E2 peptideantibody inhibits HIV.

Example 6 HIV Particle RIP Precipitation

To determine if the anti-E2 antibodies cross-react with HIV, theinventors infected GHOST CD4+ cells (Cecilia et al., 1998) with an R5HIV isolate, and then grew the cells in methionine free mediasupplemented with ³⁵S-methionine. Virus released into the culturesupernatant was partially purified by size-exclusion chromatography(FIG. 13). The p24 antigen positive peak represents radiolabeled HIVparticles, and SDS-PAGE demonstrated many ³⁵S-labeled proteins,including proteins with relative molecular weights of 41 kD, 120 kD, and160 kD consistent with HIV structural proteins (data not shown). The³⁵S-labeled material was incubated in normal mouse IgG overnight at 4°C., and material reacting with IgG non-specifically was removed byprecipitation with staph protein A (pansorbin). The supernatant was thenincubated with either normal mouse IgG or murine anti-GBV-C E2monoclonal antibody overnight (at various concentrations) at 4° C.Immune complexes were then precipitated using pansorbin, and thepelleted IgG-HIV complexes were washed extensively. Following washing,radiolabeled material was released by adding SDS and boiling, and cpmreleased was counted. FIG. 14 demonstrates results for M6 antibody,showing a dose-dependent precipitation of radiolabled HIV particles.Other anti-GBV-C E2 antibodies (including Biodesign, Virostat, M3, M5)immunoprecipitated HIV particles. For a positive control, a humananti-HIV monoclonal antibody and human HIV-negative antibodies were alsotested, and confirmed that anti-HIV antibodies precipitated theradiolabeled HIV particles (data not shown).

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A therapeutic composition comprising a GBV-C peptide binding agent,wherein the composition attenuates HIV infectivity.
 2. The compositionof claim 1, wherein the binding agent is an antibody.
 3. The compositionof claim 2, wherein the antibody is a human antibody.
 4. The compositionof claim 2, wherein the antibody is a monoclonal antibody.
 5. Thecomposition of claim 4, wherein the antibody is a humanized antibody. 6.The composition of claim 1, wherein the binding agent is an aptamer. 7.The composition of claim 1, wherein the GBV-C peptide is derived from aGBV-C envelope protein.
 8. The composition of claim 7, wherein the GBV-Cenvelope protein is a E2 protein.
 9. A method for preventing or treatingHIV infection comprising administering to a subject a compositioncomprising a GBV-C peptide binding agent, wherein the binding agentattenuates HIV infectivity.
 10. The method of claim 9, wherein thebinding agent is an anti-GBV-C E2 antibody.
 11. The method of claim 10,wherein the antibody is a human antibody
 12. The method of claim 10,wherein the antibody is a monoclonal antibody.
 13. The method of claim12, wherein the antibody is a humanized antibody.
 14. The method ofclaim 9, wherein the binding agent is an aptamer.
 15. The method ofclaim 9, further comprising administration of at least a second anti-HIVtherapy.
 16. The method of claim 15, wherein the second anti-HIV therapyis administration of an infectious GBV-C virus.
 17. The method of claim16, wherein the GBV-C virus is administered before the therapeuticcomposition.
 18. The method of claim 15, wherein the second anti-HIVtherapy is HAART therapy.
 19. The method of claim 15, wherein the secondanti-HIV therapy is AZT therapy.
 20. The method of claim 9, wherein thetherapeutic composition is administered at least twice.
 21. A method ofpreparing an antibody comprising immunizing a non-human animal with aGBV-C E2 protein or fragment thereof.
 22. A method of preparing atherapeutic composition comprising: a) contacting a cell with apolynucleotide encoding a HIV attenuating GBV-C peptide binding agentunder conditions effective to allow expression of all or part of a GBV-Cpeptide binding agent; b) collecting the expressed GBV-C peptide bindingagent; and c) constituting the GBV-C peptide binding agent in apharmaceutically acceptable solution.
 23. The method of claim 22,wherein the GBV-C peptide binding agent is an antibody.
 24. The methodof claim 23, wherein the antibody is a human antibody.
 25. The method ofclaim 23, wherein the anibody is a monoclonal antibody.
 26. The methodof claim 25, wherein the antibody is a humanized antibody.
 27. Thecomposition of claim 22, wherein the binding agent is an aptamer.
 28. Avaccine comprising an antigen derived from a GBV-C polypeptide.
 29. Thevaccine of claim 28, wherein the antigen is all or part of a GBV-C E2polypeptide.
 30. The vaccine of claim 29, wherein the antigen is a GBV-CE2-derived peptide.
 31. The vaccine of claim 30, wherein the peptide hasan amino acid sequence comprising FYEPLVRRC (SEQ ID NO: 8).
 32. Thevaccine of claim 31, wherein the peptide has an amino acid sequencecomprising LTGGFYEPLVRRC (SEQ ID NO:6).
 33. The vaccine of claim 32,wherein the peptide has an amino acid sequence comprisingGGAGLTGGFYEPLVRRC (SEQ ID NO:7).
 34. A method of immunizing a subjectcomprising contacting said subject with a composition comprising a GBV-Cpolypeptide or fragment thereof.
 35. The method of claim 35, whereinsaid composition further comprises an adjuvant.
 36. The method of claim35, wherein said GBV-C polypeptide is an E2 polypeptide.
 37. The methodof claim 36, wherein the E2-polypeptide is an E2 peptide.
 38. The methodof claim 37, wherein the E2 peptide has an amino acid sequencecomprising FYEPLVRRC (SEQ ID NO:8).
 39. The method of claim 38, whereinthe E2 peptide has an amino acid sequence comprising LTGGFYEPLVRRC (SEQID NO:6).
 40. The method of claim 39, wherein the E2 peptide has anamino acid sequence comprising GGAGLTGGFYEPLVRRC (SEQ ID NO:7).